Skip to main content

Full text of "Annual report : National Institute of Child Health and Human Development"

, »^V \ ^/ \ *^"/ \ ^-^z \ ^*^V \ ^/ % 



*, 







^ ^. % 



\ v^^ 



^-i^^ofo 



^^^. V^ 



.t^^'%. 



i;' v' 









^. 



^ ^. 



\ %,V° ^/ \ *..^v<^ /- \ 



w>i 



'<^^ 



.-^ 



-.'•'^Oi 



\ .^^ 



.•r 



^<' 
'% 






->^ V 






%. 






\ #" /^^, \ 4 



,r 



9:^'%. 



\ 















m 



^ ^. 



.^ 



/ \ 



\. 



"i 



r 



.<5«W 



"^He^ 







.•0^ 



/^ 



\ 



tYEPW 



.r 



\ 



# 



%. 



.s# 









;/^ X ^^^^^^^ .^^^ X ^^^^^ /^ X ^^^^^^^ .^^^^ X "^ /^ X ^^* /^ X '^^^~ .^^^^ 






■^ / _^^t \ / .s. \ / <fi."% \ / «c 



^ 

% 



HEA'2' 



^^^''^t 






/ 



i>. ^^ 






.•^ 






<S)v/-(^"^ 



./^ 



/ % 



.■^^ 



.^ 


















.# 



^ cS" -fS>'-'* ■* 



*/®.5^ 









\^^^\^^ 



^^^ K 'V- 



^A 



"^ ^^ f^l ^ : g<^i 



\ >- 



/^ ..^-^ \. .^-^* .<s^^^ ^" 



m \ 



*0/^^° 



.^^ 



% v^" z:^:^ % v^^ cC:*. ^-^^^ 






>^ ^^^ 



% 






\ 






0/ ^^ 












•fe, 



,#^ 






.f^ 



<3^ -^ 



\ / ^-'o 



■^^ 






^-^tf^ 



^x. 






<■*. 



\ 



^(•V^'^ 









% 4 



^"K 






/i^^ 






% 4 



^"^ 



1^ A 



'YE^\> 



.# 



.#^ 






^1? v^ 



/" ^^^u^^ \ ^/^ ^'^^'^ 



,, y "^^ 



Co 






im 



^^'"o. 









'% 






,^^ x;"^"./ x,'°^r.#^'^ x;*^.-^^^ X ^"^■^*\.^^^ \/^^^#^^ x "'^r.< 



:t^ 



.^^ 






^^''^ 



\ v^ 






.^ 



'^ \- % 



% 



.^^ 






^^* 






.^^^ 






.^i., (llj 









WEf 



■yj. 






\ *«^ </ \. ^' 







.# 







NATIONAL INSTITUTES OF H. 

r 

ULU JO i 



National Institute of Child Health and Human Development 



NICHD 

ANNUAL REPORT 

FY95 

October 1, 1994 to September 30, 1995 

PART 1 



phi 



PROJECT NUMBER LISTING 

for the 

ANNUAL REPORT 

of the 

NICHD 



October 1, 1994 - September 30, 1995 



Project Numbers - Annual Report 1995 



BIOMETRY BRANCH (BB) 



ZOl HD 00841-13 
TERMINATED 



Methods for Comparing and Analyzing Data from Several Complex Surveys 
Kai F. Yu, Ph.D. 



ZOl HD 00855-04 



Statistical Methods for the Common Odds Ratio of a Number of Contingency Tables 
Kai F. Yu, Ph.D. 



ZOl HD 00856-04 



Statistical Methods for a Mixture of Subpopulations 
Kai F. Yu, Ph.D. 



ZOl HD 00857-04 



Methodological Research in Mathematical Statistics and Biostatistics 
Young J. Lee, Ph.D. 



ZOl HD 00859-04 



Meta-Analytic Methods 

Rebecca DerSimonian, Ph.D. 



ZOl HD 00864. 00866-869 



Project Numbers - Annual Report 1995 

CELL BIOLOGY AND METABOLISM BRANCH (CBMB) 

ZOl HD 01600-1 1 Biochemical Basis of T Cell Activation 

Larry E. Samelson, M.D. 

ZOl HD 1 602- 1 1 Regulation of Intracellular Iron Metabolism 

Tracey A. Rouault, M.D. 

ZOl HD 01606-07 The Biology of Early Organelles of the Secretory Pathway 

Richard D. Klausner, M.D. 

ZO 1 HD 1 607-05 Protein Trafficking in the Secretory Pathway 

Juan Bonifacino, Ph.D. 

ZOl HD 01608-05 Gene Regulation in Response to Environmental Stress 

Gisela Storz, Ph.D. 

ZO 1 HD 1 609-04 Localization and Dynamics of Intracellular Organelles 

Jennifer Lippincott-Schwartz, Ph.D. 

ZO 1 HD 1 6 1 0-03 Intracellular Metal Metabolism 

Richard D. Klausner, M.D. 

ZO 1 HD 1 6 1 1 -0 1 The VHL Tumor Suppressor Gene 

Richard D. Klausner, M.D. 

ZOl HD 01612-99 



Project Numbers - Annual Report 1995 

COMPUTER SCIENCES BRANCH (CSB) 

ZO 1 HD 1 802-05 Data Coordinating Center for the NICHD Study of Early Child Care 

Bonnie K. Knoke 



Project Numbers - Annual Report 1995 

DEVELOPMENTAL ENDOCRINOLOGY BRANCH (DEB) 



ZOl HD 00610-15 



ZOl HD 00615-15 



ZOl HD 00616-14 
TERMINATED 

ZOl HD 00618-14 



ZOl HD 00623-12 



ZOl HD 00627-06 



Growth, Puberty, Their Disorders: Physiology, Pathophysiology, and Molecular Biology 
Gordon B. Cutler, Jr., M.D. 

Endocrine-Immune Interactions 

George P. Chrousos, M.D. 

Structure, Function, and Physiology of Glycoprotein Hormones 
Bruce C. Nisula, M.D. 

Physiology and Pathophysiology of the Hypothalamic-Pituitary-Adrenal Axis 
George P. Chrousos, M.D. 

Adrenal Physiology, Pathophysiology, and Molecular Biology 
Gordon B. Cutler, Jr., M.D. 

Glycoprotein Hormones: Oligosaccharide Structure and Function 
Diana Blithe, Ph.D. 



ZOl HD 00628-06 



ZOl HD 00631-06 



ZOl HD 00632-06 



Biological Roles and Mechanisms of Action of Insulin-Like Growth Factors (IGFs) 
Carolyn Bondy, M.D. 

Neuroendocrine Control of the Stress Reponse 
Greti Aguilera, M.D. 

Physiological Actions of the Renin- Angiotension-System 
Greti Aguilera, M.D. 



ZOl HD 00633-05 



ZOl HD 00634-03 
TERMINATED 

ZOl HD 00636-03 



ZOl HD 00637-02 



ZOl HD 00638-02 



Ovarian Folliculogenesis 

Lawrence Nelson, M.D. 

Metabolic Effects of Insulin-like Growth Factor I in Normal and Diabetic Adolescents 
Carolyn Bondy, M.D. 

Endocrinology of Reproduction in Women 
James Segars, M.D. 

Steroid Hormone Action in Female Reproduction 
Lyrmette Nieman, M.D. 

Physiology of Hypercortisolism 
Lyrmette Nieman, M.D. 



ZOl HD 00639-99 



Project Numbers - Annual Report 1995 

DIVISION OF EPIDEMIOLOGY, STATISTICS AND PREVENTION RESEARCH (DESPR) 

ZO 1 HD 1 703-06 Study of Pregnancy Outcome, Maternal Death and Child Health in Pakistan 

Heinz W. Berendes, M.D., M.H.S. 

ZO 1 HD 1 704-05 Stunting Among Bedouin Arab Children in the Negev, Israel 

PRJT. COMPLETED (10/1-31/94) Heinz W. Berendes, M.D., M.H.S. 

ZO 1 HD 1 705-02 Women's Lifestyles in Pregnancy Study, Analysis of Data 

PRJT. COMPLETED (10/1-12/1/94) Heinz W. Berendes, M.D., M.H.S. 

ZO 1 HD 1 706-0 1 A Randomized Controlled Trial for the Evaluation of a New Antenatal Care Model 

Heinz W. Berendes, M.D., M.H.S. 

ZOl HD 01707-99 



Project Numbers - Annual Report 1995 

ENDOCRINOLOGY AND REPRODUCTION RESEARCH BRANCH (ERRB) 

ZOl HD 00035-23 



ZOl HD 00146-19 
TERMINATED 



The Structure and Function of Peptides and Proteins 
Hao-Chia Chen, Ph.D. 

Structural Studies of Proteins 
Hao-Chia Chen, Ph.D. 



ZOl HD 00147-19 
TERMINATED 

ZOl HD 00150-20 



ZOl HD 00184-17 



ZOl HD 00187-16 



ZOl HD 00193-10 



ZOl HD 00194-07 



ZOl HD 00195-02 



Mechanism of Action of Peptide Hormones in Steroidogenic Cells 
Maria L. Dufau, M.D., Ph.D. 

Characterization of Gonadal Receptors and Mechanisms of Action of Peptide Hormones 
in Steroidogenic Cells 

Maria L. Dufau, M.D., Ph.D. 

Regulation of Pituitary Hormone Secretion 
Kevin J. Catt, M.D., Ph.D. 

Hormonal Regulation of Cellular Metabolism 
Kuo-Ping Huang, Ph.D. 

Angiotensin II Receptors and Activation Mechanisms 
Kevin J. Catt, M.D., Ph.D. 

Steroid Biosynthesis and Metabolism in the Mammalian Adrenal Cortex 
Charles A. Strott, M.D. 

Intracellular Signalling in Endocrine Cells 
Stanko Stojilkovic, Ph.D. 



ZOl HD 00196-301 



Project Numbers - Annual Report 1995 



ZOIHD 00331-12 



EPIDEMIOLOGY BRANCH (EB) 

Diabetes in Early Pregnancy (DIEP) 

James L. Mills, M.D., M.S. 



ZOl HD 00334-12 



Low Birth Weight Across Generations 

Mark A. Klebanoff, M.D., M.P.H. 



ZOl HD 00361-09 



ZOl HD 00369-07 



Child Health Supplement to the 1988 National Health Interview Survey 
Mary D. Overpeck, M.P.H. 

Adverse Perinatal Events and Subsequent Injury-related Death 
Mark A. Klebanoff, M.D., M.P.H. 



ZOl HD 00373-07 



Calcium Supplementation in Pregnancy for the Prevention of Preeclampsia 
Richard J. Levine, M.D. 



ZOl HD 00379-06 



Data Analysis from the Vaginal Infections and Prematurity Study 
Mark A. Klebanoff, M.D., M.P.H. 



ZOl HD 00382-05 Cocaine and Marijuana Use During Pregnancy and Pregnancy Outcome 

PRJT. COMPLETED (10/1/94-2/28/95) Mark A. Klebanoff, M.D., M.P.H. 



ZOl HD 00383-05 



Analyses of Data from the Collaborative Perinatal Project 
Mark A. Klebanoff, M.D., M.P.H. 



ZOl HD 00384-04 



Field Trial of Oral Cholera Vaccines in Bangladesh 
John D. Clemens, M.D. 



ZOl HD 00385-04 



Epidemiology of Rotavirus Infections in Bangladesh 
John D. Clemens, M.D. 



ZOl HD 00386-04 



ZOl HD 00389-04 



Evaluation of a Water-Sanitation Intervention in Egypt 
John D. Clemens, M.D. 

Studies of the Epidemiology of Pediafric Shigellosis in Bangladesh 
John D. Clemens, M.D. 



ZOl HD 00392-04 



Fatal Injuries to U.S. Infants, 1983-87 

Ruth A. Brenner, M.D., M.P.H. 



ZOl HD 00393-04 Trends in Death Rates from Drowning Among Children, 1971-88 

PROJ. COMPLETED (10/1/94-5/31/95) Ruth Brenner, M.D., M.P.H. 



ZOl HD 00801-20 



Studies Based on the Medical Birth Registries of Norway and Sweden 
Allen A. Herman, M.D., Ph.D. 



ZOl HD 00861-13 



Assessment of In-Utero Fetal Growth Patterns in Relation to Outcome at Birth 
Allen A. Herman, M.D., Ph.D. 



Continued - 



Project Numbers - Annual Report 1995 

EPIDEMIOLOGY BRANCH (EB) 

(continued) 

ZOI HD 00872-10 Factors Associated with Premature Births: Missouri Follow-Back 

Survey 

Allen A. Herman, M.D., Ph.D. 

ZOI HD 2500-03 ETEC Seroepidemiology Evaluations in Alexandria, Egypt 

John D. Clemens, M.D. 

ZO 1 HD 2502-03 NICHD-Health Research Board of Ireland Neural Tube Defects Study 

James L. Mills, M.D., M.S. 

ZOI HD 2503-03 1991 Follow-up National Maternal and Infant Health Survey 

PROJ. COMPLETED (10/1/94-3/31/95) Mary D. Overpeck, M.P.H. 

ZOI HD 2505-03 Infectious Disease Mortality During Infancy: United States, 1987 

PROJ. COMPLETED (10/1-12/31/94) Jennifer S. Read, M.D., M.P.H. 

ZOI HD 2506-03 Lack of Age- Appropriate Immunizations Among Infants Bom in D.C. 

Ruth A. Brenner, M.D., M.P.H. 

ZOI HD 2507-03 Prostaglandin Excretion in Preeclampsia 

James L. Mills, M.D., M.S. 

ZO 1 HD 25 1 0-03 Diet, Maternal Nutritional Status, Blood Pressure and Fetal Growth 

Nebiat Tafari, M.D. 

ZOI HD 251 1-02 Immunogenecity of Routine Childhood Vaccines in HIV Positive Children 

Jennifer S. Read, M.D. 

ZO 1 HD 25 12-02 Birth Certificate Linkage to Growth and Health Measures Using NHANES III 

Mary D. Overpeck, M.P.H. 

ZOI HD 25 13-02 High Status and Risk Factors Analysis of Premature High Risk Deliveries 

Mary D. Overpeck, M.P.H. 

ZOI HD 2514-02 Childhood Drowning Deaths - A National Analysis of Death Certificate Information 

Ruth A. Brenner, M.D., M.P.H. 

ZO 1 HD 25 1 5-02 Prevention of Childhood Injuries - Phase I Injury Surveillance 

Ruth A. Brenner, M.D., M.P.H. 

ZO 1 HD 2 5 1 6-02 The Role of Bathtub Rings and Seats in Infant Drowning Deaths 

Ruth A. Brenner, M.D., M.P.H. 

ZO I HD 25 1 7-02 First Week Changes in Birthweight and Other Neonatal Anthropometric Parameters 

Nebiat Tafari, M.D. 

Continued 



Project Numbers - Annual Report 1 995 

EProEMIOLOGY BRANCH (EB) 

(continued) 

ZOl HD 2518-02 Effectiveness of Rapid Plasma Reagin Screening in Gestational Syphillis 

Nebiat Tafari, M.D. 

ZO 1 HD 2519-01 Determination of Protection Level of Maternal Antibody to Group B Streptococcus 

Feng-Ying Lin, M.D., M.P.H. 

ZO 1 HD 2520-0 1 Maternal Caffeine Use in Pregnancy Outcome 

Mark A. Klebanoff, M.D., M.P.H. 

ZOl HD 2521-01 Replacement of Pregnancy Losses: Interpregnancy Interval in Adolescence 

Yvette R. Johnson, M.D., M.P.H. 

ZOl HD 2522-01 Intrapartum Antibiotic Prophylaxis Against Early-Onset Group B Streptococcal Disease 

Yvette R. Johnson, M.D., M.P.H. 

ZOl HD 2523-01 Studies Based on Maternally Linked Birth Registries of Missouri and Utah 

Allen A. Herman, M.D., Ph.D. 

ZO 1 HD 2524-0 1 Occupational Nonfatal Injuries in U.S. Children 

Mary D. Overpeck, M.P.H. 

ZOl HD 2525-0 1 World Health Organization Study of Health Behavior in School Children (WHO-HBSC) 

Mary D. Overpeck, M.P.H. 



ZOl HD 002526-2600 



Project Numbers - Annual Report 1 995 

HERITABLE DISORDERS BRANCH (HDB) 



ZOl HD 00131-21 



ZOI HD 00137-21 



ZOl HD 00404-13 



ZOl HD 00408-12 



ZOl HD 00415-04 
(Merged with 
ZOl HD 00910-16) 

ZOl HD 00417-04 
TERMINATED 

ZOl HD 00910-16 



ZOl HD 00912-16 



Human Biochemical Genetics 

William A. Gahl, M.D., Ph.D. 

Regulation and Expression of the UDP-Glucuronosyltransferase Gene Family 
Ida S. Owens, Ph.D. 

Cell and Sulfur Metabolism in Fibroblasts of Genetic Diseases 
Jean DeBrohun Butler, Ph.D. 

Heritable Disorders of Connective Tissue 
Joan C. Marini, M.D., Ph.D. 

Physiology and Molecular Biology of Arachidonate Metabolism and CFTR Mutation 
inCF 

Anil B. Mukherjee, M.D., Ph.D. 

Molecular Basis of alpha 1 -Antitrypsin Deficiency 
Mark Brantly, M.D. 

Study of Uteroglobin, Phospholipase A,, and Osteopontin Genes 
Anil B. Mukherjee, M.D., Ph.D. 

Gene Regulation and Cellular Differentiation 
Janice Y. Chou, Ph.D. 



ZOl HD 00139-45 

then use 00418-499 



Project Numbers - Annual Report 1995 

LABORATORY OF COMPARATIVE ETHOLOGY (LCE) 

ZO 1 HD 1 1 06- 1 2 Developmental Continuity of Individual Differences in Reactivity in Monkeys 

Stephen J. Suomi, Ph.D. 

ZOl HD 01 107-12 Adaptation of Laboratory Reared Monkeys to Field Environments 

Stephen J. Suomi, Ph.D. 

ZO 1 HD 1 1 1 2-09 Effects of Home and Out-of-Home Care on Child Development 

Michael E. Lamb, Ph.D. 

ZOl HD Oil 14-08 Individual Differences in Physical and Affective Functioning in Infancy 

Michael E. Lamb, Ph.D. 

ZOl HD 01 1 15-08 Effects of Domestic Violence and Evaluation of Children's Testimony 

Michael E. Lamb, Ph.D. 

ZO 1 HD 0111 6-08 Social Development Across the Lifespan in Diverse Cultures and Ecologies 

Michael E. Lamb, Ph.D. 

ZO 1 HD 0111 7-08 The Hospitalization Experience: Children's Coping with the Stress of 

Surgery 

Marc H. Bomstein, Ph.D. 

ZO 1 HD 1 1 1 9-08 Specificity of Mother-Infant Interaction 

Marc H. Bomstein, Ph.D. 

ZOl HD 01 120-08 Parenting and Infancy Activity: A Multiculture Perspective 

Marc H. Bomstein, Ph.D. 

ZOl HD 01 122-08 Longitudinal Assessment of Children's Mental and Social Abilities 

Marc H. Bomstein, Ph.D. 

ZOl HD 01 123-05 Physiological Correlates and Neural Mechanisms of the Infant Cry and Related 

Vocalizations 

John Newman, Ph.D. 

ZOl HD 01 124-05 Genetic and Experiential Influences on the Development of Primate Vocal Behavior 

John Newman, Ph.D. 

ZOl HD 01 125-02 Psychophysiological Substrates of Cognitive Processing and Socioemotional Expression 

TERMINATED from 2 to 36 Months 

Marc H. Bomstein, Ph.D. 



ZOl HD 01126-1199 



Project Numbers - Annual Report 1 995 

LABORATORY OF CELLULAR AND MOLECULAR NEUROPHYSIOLOGY (LCMN) 



ZOl HD 00707-11 



ZOl HD 01205-03 



ZOl HD 01206-02 



ZOl HD 02000-04 



Pharmacological Studies of Synaptic Transmission In Vitro 
Mark L. Mayer, Ph.D. 

Cellular and Synaptic Physiology of Hippocampal Intemeurons 
Chris J. McBain, Ph.D. 

Receptor Mediated Calcium Signalling in Glia and Neurons 
James T. Russell, D.V.M., Ph.D. 

Neurotransmitter Receptors in Glia 
Vittorio Gallo, Ph.D. 



ZOl HD 01207-99 



Project Numbers - Annual Report 1995 

LABORATORY OF DEVELOPMENTAL AND MOLECULAR IMMUNITY (LDMI) 

ZO 1 HD 01301-13 Human Immune Response to Polysaccharide-Protein Conjugate Vaccines 

Rachel Schneerson, M.D. 

ZOl HD 01308-12 Conjugate-Induced Polysaccharide Antibodies 

Shousun C. Szu, Ph.D. 

ZOl HD 0131 1-02 A Polysaccharide Vaccine for a Mycobacterium-Tuberculosis 

John B. Robbms, M.D. 

ZOl HD 1 3 1 2-0 1 Isolation and Purification of Subunit B of Shiga Toxin 

Vince Pozsgay, Ph.D. 

ZOl HD 01313-01 Synthetic Vaccine Against Shigellosis 

Vince Pozsgay, Ph.D. 

ZO 1 HD 01314-01 Analysis and Synthesis of Carbohydrate Antigens of Mycobacterium Tuberculosis 

Vince Pozsgay, Ph.D. 

ZO 1 HD 01315-01 Modulation of Protein and Cell Functions by Heparin/Heparan Sulfates 

Audrey L. Stone, Ph.D. 

ZOl HD 01316-99 



Project Numbers - Annual Report 1 995 

LABORATORY OF DEVELOPMENTAL NEUROBIOLOGY (LDN) 

ZOl HD 00047-26 



ZOl HD 00056-20 



ZOl HD 00064-19 



ZOl HD 00094-25 



ZOl HD 00095-25 



ZOl HD 00704-11 



ZOl HD 00708-11 



ZOl HD 00710-07 



ZOl HD 00711-06 



ZOl HD 00712-04 



ZOl HD 00713-01 



ZOl HD 01202-07 
TERMINATED 



Biochemical Studies of Neurons and Other Cell Types 
Douglas E. Brermeman, Ph.D. 

Biosynthesis, Processing and Secretion of Neuropeptides and Pituitary 
Peptide Hormones 

Yoke Peng Loh, Ph.D. 

Neurobiologic Studies of Neurons and Glia in Cell Culture 
Phillip G. Nelson, M.D., Ph.D. 

Pineal Regulation: Environmental and Physiology Factors 
David C. Klein, Ph.D. 

Pineal Regulation: Transsynaptic and Intracellular Mechanisms 
David C. Klein, Ph.D. 

Tetanus Toxin Effects and Localization in Neurons 
Elaine A. Neale, Ph.D. 

Morphological Studies of Neuronal and Non-Neuronal Cells in CNS Cell Cultures 
Elaine A. Neale, Ph.D. 

Molecular Characterization of Glutamate Receptor Expression in Brain 
Andres Buonarmo, Ph.D. 

Transcriptional Regulation of Skeletal Muscle-Specific Genes by Electrical Activity 
Andres Buonarmo, Ph.D. 

Regulation of Phenotypic Differentiation in the Developing Mammalian CNS 
Denes Agoston, M.D. 

Regulation of Gene Transcription and Neurite Outgrowth by Neural Impulse Activity 
Richard D. Fields, Ph.D. 

Regulation of Expression and Function of Neuropeptides During Development 
Yoke Peng Loh, Ph.D. 



ZOl HD 00714-799 



Project Numbers - Annual Report 1995 

LABORATORY OF EUCARYOTIC GENE REGULATION (LEGR) 



ZOl HD 01004-12 



ZOl HD 01009-03 



ZOl HD 01010-01 



Regulation of Amino Acid and Nucleotide Biosynthesis in Saccharomyces 
Cerevisiae 

Alan G. Hinnebusch, Ph.D. 

Regulation and Function of Genetic Elements 
Henry Levin, Ph.D. 

Regulation of Eukaryotic Protein Synthesis 
Thomas E. Dever, Ph.D. 



Above project numbers are also included in the FY95 listing for the Laboratory of Molecular Genetics (LMG). 



Project Numbers - Annual Report 1995 

LABORATORY OF MOLECULAR EMBRYOLOGY (LME) 

ZOl HD 01900-05 Developmental Regulation of Differential Gene Expression 

Alan Wolffe, M.D. 

ZO 1 HD 01901-01 Gene Regulation by Thyroid Hormone during Tissue Remodeling 

Yun-Bo Shi, Ph.D. 

ZO 1 HD 1 902-0 1 Analysis of the S Phase Checkpoint in Higher Eukaryotes 

Mary Dasso, Ph.D. 

ZOl HD 01903-99 



Project Numbers - Annual Report 1995 

LABORATORY OF MOLECULAR GENETICS (LMG) 



ZOl HD 00066-25 



ZOl HD 00067-27 



ZOl HD 00068-24 



ZOl HD 00069-23 



ZOl HD 01002-13 



ZOl HD 01004-12 



ZOl HD 01005-08 



ZOl HD 01006-07 



ZOl HD 01008-06 



ZOl HD 01009-03 



ZOl HD 01010-01 



Control Mechanisms in Temperate Bacteriophage Lambda 
Robert A. Weisberg, Ph.D. 

The Integration of Macromolecular Synthesis in Escherichia coli 
Michael Cashel, M.D., Ph.D. 

Factors Influencing Genetics Transcription-Initiation and Termination 
Robert J. Crouch, Ph.D. 

Molecular Genetics of Mammalian Retrovirus Replication 
Judith G. Levin, Ph.D. 

Gene Expression During Embryonic Development of Xenopus Laevis 
Igor B. Dawid, Ph.D. 

Regulation of Amino Acid and Nucleotide Biosynthesis in Saccharomyces 
Cerevisiae 

Alan G. Hirmebusch, Ph.D. 

Regulation of Cellular Proliferation and Diversity in Drosophila 
James A. Kennison, Ph.D. 

Protein-Nucleic Acid Interactions in Vertebrate Embryogenesis 
Thomas D. Sargent, Ph.D. 

Molecular Genetics of Protein-Nucleic Acid Interactions in Drosophila 
Susan R. Haynes, Ph.D. 

Regulation and Function of Genetic Elements 
Henry Levin, Ph.D. 

Regulation of Eukaryotic Protein Synthesis 
Thomas E. Dever, Ph.D. 



ZOl HD 01011-99 



Project Numbers - Annual Report 1995 

LABORATORY OF MAMMALIAN GENES AND DEVELOPMENT (LMGD) 

ZOl HD 00071-23 Gene and Transgene Regulation in the Developing Mouse 

Heiner Westphal, M.D. 

ZO 1 HD 1 800-05 The Genetic Basis of Mammalian Kidney Development 

TERMINATED Gregory Dressier, Ph.D. 

ZO 1 HD 1 80 1 -06 The Molecular Analysis of Murine Development 

Kathy Mahon, Ph.D. 

ZOl HD 01803-02 Genetic Analysis of Thymocyte Development 

Paul Love, M.D. 

ZOl HD 01804-99 



Project Numbers - Annual Report 1995 

LABORATORY OF MOLECULAR GROWTH REGULATIONS (LMGR) 

ZO 1 HD 004 1 2-08 Molecular Regulation of Gene Expression 

Richard J. Maraia, M.D. 

ZO 1 HD 00505-02 Eukaryotic Transcriptional Regulation 

Yoshihiro Nakatani, Ph.D. 

ZOl HD 013 10-09 Developmental Gene Regulation of the Immune System 

Keiko Ozato, Ph.D. 

ZOl HD 08719-15 Development and Uses of Eukaryotic Vectors 

Bruce H. Howard, M.D. 

ZOl HD 00506-599 



Project Numbers - Annual Report 1995 

LABORATORY OF THEORETICAL AND PHYSICAL BIOLOGY (LTPB) 



ZOl HD 00165-20 



ZOl HD 00171-19 



ZOl HD 01400-13 



ZOl HD 01408-04 
TERMINATED 

ZOl HD 01409-10 



ZOl HD 01415-05 



ZOl HD 01416-03 



ZOl HD 01417-02 



ZOl HD 01418-02 



ZOl HD 01419-02 



ZOl HD 01420-01 



ZOl HD 01501-04 



Isolation and Characterization of Macromolecular and Cellular Particles 
Andreas C. Chrambach, Ph.D. 

Electrophoretic Methodology 

Andreas C. Chrambach, Ph.D. 

Dynamics of the Growth and Development of Bone 
Alfred L. Yergey, Ph.D. 

Stability and Specificity of DNA-Protein Interactions 
Mark M. Gamer, Ph.D. 

Membrane Transport and Fusion 

Joshua Zimmerberg, M.D., Ph.D. 

Kinetics of Exocytosis 

Joshua Zimmerberg, M.D., Ph.D. 

Tissue Imaging in Cell Biology 
Leonid Margolis, Ph.D. 

Energetics of the Interaction Between Water, Membranes, and Macromolecules 
Alfred L. Yergey, Ph.D. 

Regulation of Mitosis at the Cellular Level 
Frank A. Suprynowicz, Ph.D. 

Localization of Membrane Fusion Proteins 
Joshua Zimmerberg, M.D., Ph.D. 

Calcium Triggered Membrane Trafficking in Sea Urchin Eggs 
Steven Vogel, Ph.D. 

Role of Lipids in Membrane Rearrangements 
Leonid V. Chemomordik, Ph.D. 



ZOl HD 01421-99 



Project Numbers - Annual Report 1 995 

OFFICE OF THE SCIENTIFIC DIRECTOR (OSD) 

ZOl HD 00093-21 Mechanism of Action of Nerve Growth Factor 

Gordon Guroff, Ph.D. 

ZOl HD 01500-13 Studies on DNA Replication, Repair, and Mutagenesis in Eukaryotic and 

Prokaryotic Cells 

Arthur S. Levine, M.D. 

ZOl HD 01502-99 



Project Numbers - Annual Report 1995 

PERINATOLOGY BRANCH (PB) 

ZOl HD 02400-04 The Role of Subclinical Infection and Cytokines in Preterm Parturition 

Roberto Romero, M.D. 

ZOl HD 02401-03 Prenatal Diagnosis of Congenital Anomalies 

Roberto Romero, M.D. 

ZOl HD 2402-99 



Project Numbers - Annual Report 1 995 

PREVENTION RESEARCH BRANCH (PRB), DESPR 



ZOl HD 00876-05 



Determinants of Childhood Poison Ingestion 
Lois A. Maiman, Ph.D. 



ZOl HD 00877-05 



Prevention and Education in Coronary Heart Disease Reduction 
Lois A. Maiman, Ph.D. 



ZOl HD 2100-04 



Prenatal Care Utilization and Adverse Health Behaviors During Pregnancy 
Lois A. Maiman, Ph.D. 



ZOl HD 2101-04 



Increasing Preventive Actions for Childhood Injury Among Preschool Children 
Lois A. Maiman, Ph.D. 



ZO 1 HD 2 1 07-04 Children and Adolescent Trial of Cardiovascular Health (CATCH) 

PRJT. COMPLETED (10/1/94-8/1/95) Bruce G. Simons-Morton, Ed.D., M.P.H. 

ZOl HD 2108-04 Afro- American Adolescent Girls Growth and Development 

PRJT. COMPLETED (10/1/94-7/1/95) Bruce G. Simons-Morton, Ed.D., M.P.H. 

ZO 1 HD 2 1 09-04 How Much Physical Activity Do Children Obtain During Physical Education 

PRJT. COMPLETED (10/1/94-4/1/95) Classes? 

Bruce G. Simons-Morton, Ed.D., M.P.H. 



ZOl HD 21 10-04 



Prevention of Problem Behaviors Among Middle School Students 
Bruce G. Simons-Morton, Ed.D., M.P.H. 



ZOl HD 21 14-03 



MD/Family Partnership: Education in Asthma Self-Management 
Lois A. Maiman, Ph.D. 



ZOl HD 21 15-03 



Immunization Among D.C. Infants 

Bruce G. Simons-Morton, Ed.D., M.P.H. 



ZOl HD 21 16-03 



Preventing Pregnancies Among Adolescent Girls in the District of Columbia 
Bruce G. Simons-Morton, Ed.D., M.P.H. 



ZOl HD 21 17-02 Validity of the Physical Activity Interview and Caltrac Motion Sensor 

PROJ. COMPLETED (10/1/94-6/15/95) Bruce G. Simons-Morton, Ed.D., M.P.H. 

ZO 1 HD 2 1 1 8-0 1 Parent-Child Conflict During the Transition to Middle School 

PROJ. NEVER INITIATED-(CANCEL) Barbara Radziszewska, Ph.D.; Denise Haynie, Ph.D. 



ZOl HD 21 19-02 



Parenting Style and Adolescent Substance Use and Depressive Symptoms 
Barbara Radziszewska, Ph.D. 



ZOl HD 2120-01 
(5/2-9/30/95) 

ZOl HD2121-01 
(5/1-9/30/95) 



A Survey of Parent Intervention Strategies to Prevent Adolescent Alcohol Use 
Bruce G. Simons-Morton, Ed.D., M.P.H. 



Parent-Child Interaction and Asthma Self-Management 



Lois A. Maiman, Ph.D. 



ZOl HD 2 122-0 1 
(6/1-9/30/95) 

ZOl HD 2123-99 



Perceptions of Parents Regarding the Onset of Sexual Intercourse Among Youth 
Aria D. Crump, Sc.D. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl-HD-01600-11 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT ISO characters or less. Title must fit on one line between the borders.) 

Biochemical Basis of T Cell Activation 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation} 

PI: L.E. Samelson, Chief, SLS, CBMB 
Others: J. Donovan, IRTA, CBMB 

R. Guitian, Pre- IRTA, CBMB 

N. Isakov, Adjunct Scientist, CBMB 

Y. Ota, Visiting Fellow, CBMB 

R.L. Wange, IRTA, CBMB 



COOPERATING UNITS lif any) 

R.N. Germain and D.I. Cohen, Laboratory of Immunology, NIAID; R. Aebersold and 
J. Watts, University of Washington; W. Langdon, University of W. Australia 



LAB/BRANCH 

Cell Biology and Metabolism Branch 



SECTION 

Section on Lymphocyte Signaling 



INSTITUTE AND LOCATION 

NICHD, NIH 



TOTAL STAFF YEARS: 
6.0 



PROFESSIONAL: 
5.0 



OTHER: 
1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects D (b) Human tissues H (c) Neither 
D (a1) Minors 
D (a2) Interviews 



SUMMARY OF WORK {Use standard unreduced type. Do not exceed the space provided.) 

Engagement of the multicomponent antigen receptor in T cells (TCR) results in rapid 
activation of a protein tyrosine kinase pathway. A major TCR- associated protein 
tyrosine kinase is ZAP- 70, a protein that binds to the activated TCR. Under 
conditions of TCR crosslinking with anti-receptor antibodies, the ZAP-70 bound to 
the TCR is itself tyrosine phosphorylated and activated. Studies using T cell 
clones responding to peptide antigens demonstrate that ZAP-70 activation also 
occurs in that setting. For a given T cell clone, slight alteration of the peptide 
antigen or the major histocompatibility-encoded presenting molecule can result in 
partial activation or inhibition. Under these conditions, the TCR is partially 
tyrosine phosphorylated, and though the ZAP-70 molecule is bound to the TCR, it is 
neither tyrosine phosphorylated nor active. These studies have great relevance for 
understanding immunological tolerance as well as T cell activation. Moreover they 
demonstrate that regulation of ZAP-7 activity is at the level of phosphorylation 
as well as TCR binding. We have determined the sites of tyrosine phosphorylation of 
the ZAP-70 kinase. Using site-directed mutagenesis we have mutated two critical 
tyrosine residues in the kinase domain of ZAP-70, and have demonstrated that one 
of these sites is required for activation of the enzyme. These studies enhance our 
understanding of the mechanism of activation of this enzyme. Additional studies 
focus on a substrate of tyrosine kinases in T cells, pl20'"'-', a proto- oncogene, 
which may be involved in the ras pathway in T cells. Recent studies demonstrate 
that this protein can be found in a complex with the linker protein Grb2 . The 
amount of Grb2-pl2 0^" complex is regulated by TCR activation. Studies on the 
dysregulation of tyrosine phosphorylation pathways seen with HIV infection have 
demonstrated that the tyrosine kinases Lck and Fyn, but not ZAP-70 are activated by 
HIV gpl2 binding to CD4 . 



PHS 6040 (Rev. 5/92) 



ZOl-HD-01600-11 

BIOCHEMICAL BASIS OF T CELL ACTIVATION 

Project Description 

Objectives : 

The T cell antigen receptor (TCR) is a multicomponent structure 
that is coupled to several signal transduction pathways. Our 
earlier studies have demonstrated that receptor occupancy results 
in activation of tyrosine and serine kinases. Our current focus 
is the complete characterization of the protein tyrosine kinase 
pathway. The major goal of the unit is to identify and 
characterize the responsible kinase (s) and major substrates of 
these kinases. Only by this means will we be able to understand 
the function of this pathway and the manner by which it is 
activated and regulated. 

Methods Employed: 

Our studies of T cell activation use antigen specific murine T 
cell hybridomas, human T cell tumors, normal human peripheral T 
cells and murine thymocytes. Activation is induced either by 
specific antigen or stimulatory monoclonal antibodies. Analysis 
of signal transduction mechanisms depends on metabolic 
radiolabeling, detergent solubilization, immunoprecipitation and 
polyacrylamide gel electrophoresis . Phosphopeptide mapping has 
been performed to define sites of protein phosphorylation. 
Immunoblotting is used to detect antigen receptor subunits and 
tyrosine phosphorylated proteins. Immune complex kinase assays 
are used to determine protein tyrosine kinase activity in vitro. 
Protein tyrosine kinase domains are expressed in bacteria in 
order to obtain sufficient material for binding studies. Kinase 
protein is prepared using baculovirus expression systems. 
Phosphopeptides of defined sequence are synthesized to order. 
Kinase substrates have been purified by immunoaf f inity 
chromatography, amino acid sequence has been obtained, and the 
cDNA encoding a protein tyrosine kinase substrate has been 
cloned. Mutated forms of this substrate have been generated 
using standard site-directed techniques. Eukaryotic expression 
vectors have been used to enable expression of substrates in T 
cells. Flow cytometry is used to characterized cell cycle 
progression. 

The T Cell Receptor Tyrosine Kinase (s) : 

Identifying the kinase (s) responsible for tyrosine 
phosphorylation of the TCR and the other cellular substrates has 
been a major goal of this Section for many years. A critical 
tyrosine kinase activated by the T cell receptor is the ZAP-70 
protein, which is tyrosine phosphorylated and associates with 
the TCR upon activation. Interesting features of this protein are 



ZOl-HD-01600-11 

the tandem SH2 domains adjacent to the kinase domain. SH2 domains 
have been shown to bind proteins containing phosphotyrosine in 
other systems. In earlier studies we demonstrated that these two 
SH2 modules are required for binding to tyrosine phosphorylated 
TCR subunits. Well defined motifs in the tails of the CD3 and 
TCR^ chains, the immunoreceptor tyrosine based activation motifs 
(ITAM) , become tyrosine phosphorylated upon TCR engagement, and 
serve as the sites to which the tandem SH2 domains bind. 

A series of experiments using normal T cell clones revealed that 
ZAP-7 binding to the TCR ITAMs is insufficient for enzyme 
activation. T cell clones can be activated to produce various 
lymphokines and proliferate following TCR engagement by defined 
peptides and major histocompatibility complex (MHC) molecules. 
However over the past several years it has become clear that 
slight alterations in this complex ligand, either by subtle 
changes in the peptide or the MHC molecule, can result in an 
altered response. Partial activation in which only certain 
lymphokines are produced or complete inhibition can occur. These 
variant ligands serve as models for ligands that normally might 
regulate T cell development in the thymus, induce anergy or 
determine the response to pathogens. Our studies using normal 
clones began with the demonstration that a ligand that fully 
activates the T cells results in TCR tyrosine phosphorylation, 
ZAP-7 binding and activation with tyrosine phosphorylation of 
intracellular substrates. These results were similar to that seen 
using the Jurkat T cell tumor line and TCR crosslinking by 
antibody. The response of these T cell clones to altered ligand, 
however, was dramatically different. An unusual form of TCR 
tyrosine phosphorylation was detected in which only partial TCR 
chain tyrosine phosphorylation occurred. ZAP-7 binding to this 
subunit was detected, but this molecule was not tyrosine 
phosphorylated and was not active. Only minimal tyrosine 
phosphorylation of intracellular substrates was detected. These 
studies are relevant for understanding T cell activation during 
thymic development and induction of anergy as mentioned. They 
also show that binding of ZAP-70 to the TCR is insufficient for 
its activation, and focus attention on the regulation of the 
kinase by phosphorylation. 

With collaborators in Vancouver and Seattle we previously mapped 
the sites of tyrosine phosphorylation of the ZAP-7 kinase. Using 
a baculoviral expression system we obtained large quantities of 
the kinase. Sites of autophosphorylation and sites that became 
phosphorylated after addition of recombinant Lck protein were 
determined by mass spectrometric analysis of ZAP-7 tryptic 
peptides. Upon determination of these sites of phosphorylation in 
vitro, it was then possible to show that tyrosine residues Y292, 
Y492 and Y493 become phosphorylated after TCR engagement of 
intact cells. Site directed mutagenesis using ZAP-70 cDNA was 
used to mutate residues Y492 and Y493 to phenylalanine. These 



ZOl-HD-01600-11 

constructs were then expressed in COS cells alone or with Lck 
constructs and ZAP-7 activity was then assayed by in vitro 
kinase reactions. Native ZAP-70 expressed in COS cells has a 
basal level of activity that is markedly increased by co- 
expression of active Lck. Mutation at Y493 results in an enzyme 
with the basal level of activity but no enhanced response to co- 
expressed Lck. Phosphorylation at this site by Lck is likely a 
critical first step in activation of the enzyme. The mutation of 
Y492 to phenylalanine gave the unexpected result of leading to a 
four-fold increase in kinase activity, which was increased even 
more with Lck co-expression. It is possible that tyrosine 
phosphorylation at this residue is normally inhibitory. However 
we favor the model that Y492 when non-phosphorylated along with 
Y493 interact with critical residues in the kinase domain to 
maintain the enzyme in an inactive state. Molecular modeling 
studies support this hypothesis. The Y492F mutation would then 
result in disinhibition leading to an active kinase. 

The Tyrosine Phosphorylation Pathway in T cells: 

In previous studies we have demonstrated that activation of the 
TCR results in phosphorylation of the TCR^ chain and multiple 
intracellular substrates. The identity of the other substrates 
that are tyrosine phosphorylated upon TCR activation has been a 
major question for this Section for some time. One substrate of 
TCR-mediated tyrosine kinase activation is the protooncogene 
pl2 0''^^. In its oncogenic form, expressed as a fusion protein 
with a retroviral gag sequence the plOO''"'^" protein transforms 
pre-B cells and is expressed in the nucleus. The pl20"^^^ protein 
is non-transforming and is entirely cytoplasmic. It is rapidly 
tyrosine phosphorylated upon T cell activation. It has been shown 
to bind a number of critical intracellular signaling proteins. We 
showed earlier that the SH3 domains of one such protein, the 
linker molecule Grb2 binds to pl2 0''^^ in vitro. More recently we 
have demonstrated that a Grb2-pl2 0''*^ complex can be found in the 
cell. Binding here too is mediated by the Grb2 N-terminal SH3 
domain. Interestingly this complex is regulated by TCR engagement 
as the complex dissociates upon TCR engagement. Studies to 
determine the function of pl20''^ are in progress. 

Abnormal tyrosine phosphorylation of intracellular substrates 
might reflect inappropriate activation of intracellular 
signalling pathways. In collaboration with Dr. D.I. Cohen, LIR, 
NIAID, we have determined that such abnormal tyrosine 
phosphorylations occur in T cells after infection with HIV. We 
have discovered that one of the prominent tyrosine phosphorylated 
substrates is p34cdc2, a regulator of mitosis at the G2/M 
boundary. Cells dying of HIV- induced killing are arrested at this 
checkpoint. We have recently demonstrated other abnormalities of 
the tyrosine phosphorylation pathway. Engagement of the CD4 
molecule by HIV gpl20 results in activation of the Lck and Fyn 



ZOl-HD-01600-11 

protein tyrosine kinases without either TCR^ chain 
phosphorylation or ZAP-7 activation. Activation of the two src- 
family kinases is independent of TCR activation. Whether this 
pattern of activation of these kinases is coupled to HIV-induced 
cell death is under investigation. 



ZOl-HD-01600-11 



Publications 



Isakov N, Wange RL, Burgess WH, Watts JD, Aebersold R, and 
Samelson LE. ZAP-70 binding specificity to T cell receptor 
tyrosine-based activation motifs: The tandem SH2 domains of ZAP- 
70 bind distinct TAMs with varying affinity. J Exp Med 
1995;181:375-380. 

Kolesnitchenko V, Wahl LM, Sunila I, Tian H, Tani Y, Hartmann D- 
P, Cossman J, Raffeld M, Orenstein J, Samelson LE, and Cohen DI . 
HIV-1 envelope-initiated G2 phase programmed cell death. Proc 
Natl Acad Sci USA 1995 (In press) . 

Madrenas J, Wange RL, Wang JL, Isakov N, Samelson LE, and Germain 
RN. ^ phosphorylation without ZAP-70 activation induced by T 
cell receptor antagonists or partial agonists. Science 
1995;267:515-518. 



Samelson LE, Donovan JA, Isakov N, Ota Y, and Wange RL. Signal 
transduction mediated by the T cell antigen receptor. Annals of 
the NY Acad Sci 1995 (In press) . 



Wange RL, Guitian R, Isakov N, Watts JD, Aebersold R, and 
Samelson LE Activating and inhibitory mutations in adjacent 
tyrosines in the kinase domain of ZAP-70. J Biol Chem 
1995;270:18730-18733 . 



Wange RL, Isakov N, Burke TR Jr, Otaka A, Roller PP, Watts JD, 
Aebersold R, and Samelson LE. Inhibition of TCR signaling in 
Jurkat T Cells by a PTPase-resistant phosphoTAM peptide analog. 
J Biol Chem 1995;270:944-948. 



Watts JD, Affolter M, Krebs DL, Wange RL, Samelson LE, and 
Aebersold R. Identification by electrospray ionization mass 
spectrometry of the sites of tyrosine phosphorylation induced in 
activated Jurkat T cells on the protein tyrosine kinase ZAP-70. 
J Biol Chem 1994;269:29520-29529. 



G 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl-HD-01602-11 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Regulation of Intracellular Iron Metabolism 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
PI:T.A. Rouault, Chief, SHIM, CBMB Co-PI:R.D. Klausner, Chief, CBMB 



Others:J. Basilion, Staff Fellow, CBMB T. 
J. Butt, Adjunct Scientist, CBMB T. 
S. Cohen, Pre-IRTA, CBMB C. 

P. DeRusso, Adjunct Scientist, CBMB 
K. Iv^ai, Adjunct Scientist, CBMB 
Hae-Yeong Kim, Adjunct Scientist, CBMB 



Land, Adjunct Scientist, CBMB 
LaVaute, Research Biol ., CBMB 
Philpott, IRTA, CBMB 



COOPERATING UNITS (if any) 

D. Stout, Scripps Research Institute; W. Burgess, American Red Cross; A. Pardi, 
University of Colorado; H. Beinert & C. Kennedy, University of Wisconsin. 



LAB/BRANCH 

Cell Biology and Metabolism Branch 



SECTION 

Section on Human Iron Metabolism 



INSTITUTE AND LOCATION 
NICHD, Bethesda, MP 



TOTAL STAFF YEARS: 

8.55 



PROFESSIONAL: 
7.75 



OTHER: 



CHECK APPROPRIATE BOX{ES) 

n (a) Human subjects 

n (a1) Minors 
n (aP) IntprvJPWR 



a (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 
This project is aimed at understanding the molecular basis of intracellular iron metabolism. The cis anc 
trans elements mediating the iron-dependent alterations in abundance of ferritin and the transferrir 
receptor have been identified and characterized in previous years in this laboratory. Iron-responsive 
elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrir 
receptor mRNA. We have cloned, expressed, and characterized two essential iron-sensing proteins, Iror 
Regulatory Protein 1 (IRPl) and Iron Regulatory Protein 2 (IRP2), formerly referred to as iron-responsive 
element binding proteins (IRE-BPs) . IRPs bind IRE ' s when iron levels are depleted, resulting in the 
inhibition of translation of ferritin mRNA and prolongation of the half-life of the transferrin receptoi 
mRNA. IRPl is an iron-sulfur protein related to mitochondrial aconitase, a Krebs cycle enzyme, and it 
functions as a cytosolic aconitase in cells that are iron replete. In cells that are iron-depleted, IRP2 
binds IREs with high affinity. Regulation of RNA binding activity involves a transition from a form of IRP2 
in which a [4Fe-4S] cluster is bound, to a form that loses both iron and aconitase activity. The [4Fe-4S] 
containing protein does not bind IREs. Controlled degradation of the iron-sulfur cluster reveals that the 
physiologically relevant form of the RNA binding protein in iron-depleted cells is apoprotein. The status 
of the cluster appears to be the key to determining whether the protein will bind RNA. Little is Icnown about 
the assembly and disassembly of iron-sulfur clusters in mammalian cells. In recent studies, we hav' 
identified mammalian genes homologous to genes that are implicated in bacterial iron-sulfur clustei 
assembly. Unlike IRPl, IRP2 is actively degraded in cells that are iron-replete. An additional exon present 
in IRP2 determines whether IRP2 is subject to degradation. This 73 amino acid exon is sufficient to confer 
upon IRPl an iron -dependent degradation phenotype. Mutagenesis of cysteines within the 73 amino acid domair 
reveals that cysteines are required for degradation, and treatment of cells with proteasome inhibitors 
reveals that the proteasome is required for degradation. In order to approach questions about the physiolog> 
of iron metabolism, IRPl has been "knocked out" in mice, using homologous recombination in embryonic cell 
lines. There is no obvious phenotype associated with loss of IRPl function, and we speculate that both IRPs 
must be absent in order to see perturbations in iron metabolism. Accordingly, creation of IRP2 "knockouts 
and double "knockouts" are underway. 



PHS 6040 (Rev. 5/92) 



ZOl-HD-01602-11 

REGULATION OF INTRACELLULAR IRON 

Project description 

Objectives 

Tine main objective of this project is to understand mammalian iron metabolism. We have cloned and 
characterized through site-directed mutagenesis the two major regulatory proteins involved in sensing iron 
levels, IRP1 and IRP2. In order to better understand their mode of regulation, iron-sulfur cluster assembly 
and disassembly is being studied in vitro. Pulse-chase studies have been used to define the iron-dependent 
degradation of IRP2 and mutagenesis is being utilized to better characterize the degradation signals. Over- 
expressed and purifed apoprotein is being used to attempt to create crystals of the apoprotein and co- 
crystals with the RNA ligand. Homologous recombination and electroporation of embryonic stem cells has 
been used to created mice that are heterozygous or homozygous for deficiency of IRP1 and/or IRP2. 

Methods Employed 

Standard techniques of molecular biology are employed including cloning, site-directed mutagenesis using 
2 step PCR methodology, and transient and stable transfection. Protein chemistry techniques include one 
and two-dimensional electrophoresis, immunoprecipitation, biosynthetic labeling, and FPLC. Gel-retardation 
and resolution of complexes is performed on native gels, and in vitro translation systems are employed. 

Major Findings 

Expression of a constitutive mutant of iron regulatory protein 1 abolishes iron homeostasis in mammalian 
cells 

Iron regulatory proteins (IRPs) are iron-sensing proteins that bind to RNA stem-loop sequences known as 
iron-responsive elements (IREs) when cells are depleted of iron. Although IRPs have been previously 
shown to bind to IREs derived from ferritin and TfR mRNAs in vitro, there has not been a direct 
demonstration of the impact of a recombinant IRP on the expression of endogenous IRE- containing 
transcripts. Modulation of binding of iron regulatory protein 1 (IRP1) to IREs occurs through the assembly 
and disassembly of an iron-sulfur cluster and consequent modification of the RNA binding site. Mutation of 
each of the cysteines thought to ligate the iron-sulfur cluster of IRP1 produces a form of the protein that 
constitutively binds RNA. We evaluated the impact of expression of C437S, a mutant of IRP1 which binds 
IREs regardless of cellular iron status, on the regulation of biosynthesis of ferritin and the transferrin 
receptor. Expression of the mutant IRP1 prevented the normal regulation of ferritin and transferrin receptor 
expression. Cells continued to synthesize and express high amounts of TfR even when cells were iron- 
replete, while synthesis of ferritin remained repressed when cells were iron-replete. Thus, a single mutant 
IRP can prevent the usual homeostatic changes in synthesis of the transferrin receptor and ferritin. Cells 
expressing the mutant protein would therefore be predicted to be unable to defend against iron-overload. 
Preliminary results have shown that cells are killed by treatment with iron when C437S is expressed, and 
we are working towards creation of a tissue culture model system for the study of iron toxicity (DeRusso et 
al., 1995). 

IRP2 is regulated by protein degradation in iron-replete cells 

Binding studies have shown that IRP2 binds to consensus IREs with high affinity and specificity similar to 
IRP1. Whereas the RNA binding activity of IRP1 is regulated by the reversible assembly and disassembly of 
an iron-sulfur cluster in an otherwise stable protein, absolute levels of IRP2 are markedly decreased by iron 
treatment in several different cell types and the decrease in protein levels can be accounted for by an 
increase in the rate of degradation of IRP2 (Samaniego et al., 1994). 



ZOl-HD-01602-11 

A sequence unique to IRP2 is required for rapid iron-dependent degradation 

IRP1 and IRP2 are highly homologous proteins with an overall sequence identity of 58%. A major 
distinguishing feature between IRP1 and IRP2 is the presence of an insertion of 73 amino acids in domain 1 
of IRP2. Cloning of the genomic fragment corresponding to this sequence has revealed that the 73 amino 
acids are encoded by a single exon. When the 73 amino acid insertion was excised by site directed 
mutagenesis from IRP2, levels of the IRP2 exon deletion mutant (IRP2-73) were no longer markedly 
decreased after treatment with iron. The IRE binding affinity remained high, with an estimated Kd of 10-50 
pM, and specificity for the consensus IRE was unchanged as judged by competition assays with unlabeled 
IREs and unrelated stem-loops. These results indicated that the fundamental tertiary structure of IRP2 was 
unchanged by excision of this exon sequence. 

In order to verify that the IRP2 exon deletion mutant (IRP2-73) accumulated because of a decrease in the 
rate of degradation, as has been shown for wild type IRP2, pulse chase experiments were performed. The 
rate of degradation of IRP2 wild type in iron-treated cells was markedly faster than the rate of degradation 
of the IRP2 specific exon deletion mutant (t1/2 of 3-6 h vs. 24h), thus supporting the hypothesis that the 
IRP2 specific exon may contain degradation signals which are activated by iron. 

Insertion of the IRP2 specific exon into IRP1 results in iron-dependent degradation of recombinant IRP1 

To assess whether the 73 amino acid IRP2-specific exon contained sufficient information to direct iron 
regulated degradation of IRP2, the sequence was cloned into IRP1 at a homologous position. When 
assayed in a pulse chase experiment in the presence of iron, the IRP1/IRP2 specific exon chimeric protein 
(IRP1+73) was much more rapidly degraded than wild-type IRP1 and the degradation rate was comparable 
to that of wild type IRP2 (t1/2 of approximately 3-6 h). Thus, the 1RP2 specific exon conferred the capacity to 
be degraded at a rate similar to that seen in intact IRP2 upon IRP1 . 

The iron-dependent degradation signal of IRP2 requires the participation of cysteines in the IRP2 specific 
exon 

Cysteines are known to be critical in ligation of the [4Fe-4S] cluster of IRP1. In that setting, the iron-sulfur 
cluster is ligated by three cysteines, C437, C503, and C506, and it is the presence of a fully assembled 
[4Fe-4S] cluster that prevents RNA binding. In addition, mutation of C437 to serine (C437S) or of all three 
cysteines 437, 503, 506 to serines results in expression of a mutant protein in which IRE binding is no 
longer regulated and in which the mutant protein is permanently fixed in the IRE binding mode. In order to 
assess whether the corresponding cysteines were required for iron-dependent degradation of IRP2 or the 
IRP1/IRP2 specific exon chimeric protein (IRPI-i-73), a series of chimeric proteins containing cysteine 
mutations were assessed. Mutation of one or several of the cysteines required for cluster ligation of IRP1 
did not interfere with iron-induced degradation of chimeric IRP1/IRP2 specific exon chimeric constructs. 
Thus, the iron sensing capability of the IRP2 insert does not depend on participation of cysteines 
homologous to previously identified cysteine ligands of the iron-sulfur cluster. 

The presence of five cysteines in the IRP2 specific exon, with the spacing C (X3o)C(X5)C(X3)C(X23)C offered 
another set of cysteines which could be assessed for their role in iron-sensing. The close physical grouping 
of the three middle cysteines of the exon is noteworthy and we questioned whether the cysteines might be 
acting in concert to sense iron levels. In order to test whether cysteines from the IRP2 specific exon were 
important in degradation, the degradation rate of the chimeric protein IRP1/IRP2 specific exon (lRPI-i-73) 
was compared to the degradation rate of a similar construct which differed only in that the middle three 
cysteines, CI 68, CI 74 and CI 78, of the IRP2 specific domain were simultaneously converted to serines by 
site-directed mutagenesis (Exon 3C-S). When the three cysteines were mutated to serines, the iRP2 
specific exon could no longer transfer the capacity for rapid iron-dependent degradation to IRP1. A pulse 



ZOl-HD-01602-11 

chase experiment revealed that the exon 3C-S mutant was stable, with an estimated t1/2 of 24 h. Gel- 
retardation assays of all mutant chimeras indicated that IRE binding activity was intact and that the tertiary 
structure was therefore fundamentally intact. Thus it appears that cysteines are critical to presentation of 
the signal for iron-dependent rapid degradation, possibly because the cysteines may be involved in direct 
ligation of iron or an iron-sulfur cluster. 

Inhibition of proteasome function in vivo prevents iron dependent degradation of IRP2 

In order to gain insight into the mechanism of degradation, iron-replete cells were treated with a variety of 
reagents known to interfere with various modes of proteolysis in cells. The peptide aldehyde MG132, a 
potent inhibitor of the 20 S subunit of the proteasome, interfered with degradation of IRP2 when ceils were 
treated with iron. To further confirm the role of the proteasome in degradation of IRP2, a more specific 
inhibitor of proteasome function, lactacystin, was used. Lactacystin inhibits proteasome function by 
covalently binding to the amino terminal threonine of the mammalian proteasome subunit X. The effects of 
lactacystin in the cell are more specific for the proteasome than are those of MG132 and we found that 
lactacystin inhibits degradation of IRP2 at a level comparable to MG132. To confirm that a decrease in 
degradation rather than a change in the level of synthesis was the cause of increased protein levels, pulse 
chase experiments were performed which confirmed that degradation was inhibited. Thus, degradation of 
IRP2 is most likely mediated by the proteasome (Iwai et al., 1995). 

Translational Repressor Activity is Equivalent and is Quantitatively Predicted by in vitro RNA Binding for Two 
IRE Binding Proteins. IRP1 and IRP2 

The existence of two IRE binding proteins which are regulated by different mechanisms has 
increased the complexity of the IRP regulatory system. Thus far, there have been no descriptions of cell 
types in which one form of IRP is expressed while the other is not. It remains unclear why there are two IRE 
binding proteins. One possibility would be that the two IRPs respond to different stimuli. Another possibility is 
that the targets of the two proteins differ. Though we have previously shown using gel retardation studies 
that the two IRPs bind with equal affinity to isolated IRE motifs derived from ferritin, eALA synthase, and the 
transferrin receptor mRNAs, these studies do not necessarily mean that in the setting of the intact transcript 
that the two proteins bind with equal affinity to target IREs, as context and the potential for protein-protein 
interactions may modify binding afifinity in vivo. 

We utilized purified recombinant IRP1 and IRP2 and compared the ability of each to repress translation of 
ferritin in vitro. We showed that equal molar amounts of IRP1 and IRP2 bind isolated IREs equally, and 
furthermore that equal binding in the gel retardation assay predicted equal impact on translation of a target 
mRNA (Kim et al., 1995). 

IRP1 is not indispensable in mice 

Homologous recombination has been used to "knockout" IRP1 in mice. Heterozygotes for the "knockout of 
IRP2 have been created. Mice lacking IRP1 only are healthy, and iron uptake and metabolism appear to be 
normal. Breeding of the mice should enable us to assess the phenotype of mice lacking the gene products 
ofbothlRPI andlRP2. 



10 



ZOl-HD-01602-11 

Publications 

DeRusso PA, Philpott CC, Iwai K, Mostowski HS, Klausner RD, and Rouault 
TA. Expression of a constitutive mutant of iron regulatory protein 1 
abolishes iron homeostasis in mammalian cells. JBC 1995;26:15451-15454. 

Iwai K, Klausner RD, and Rouault TA. Requirements for iron regulated 
degradation of the RNA binding protein, Iron Regulatory Protein 2 
(IRP2). EMBO J 1995(In press). 

Jaffrey SR, Cohen NA, Rouault TA, Klausner RD, and Solomon SH. The 
iron-responsive element binding protein: A novel target for synpatic 
actions of nitric oxide. Proc Natl Acad Sci USA 1994;91:12994-12998. 

Kim HY, Klausner RD, and Rouault TA. Translational repressor activity 
is equivalent and is quantitatively predicted by in vitro RNA binding 
for two iron-responsive element binding proteins, IRPl and IRP2 . JBC 
1995;4983-4986. 

Rouault TA, Klausner RD, and Harford JB. Translational control of 
ferritin. In: J Hershey et al, eds . Translation Regualtion. Cold 
Spring Harbor: Cold Spring Harbor Press 1995 (In press). 



.11 



DEPARTMENT OF HEALTH AND HUMAN SERVtCES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl-HD-01606-07 



PERIOD COVERED 

October 1, 1994 to September 30, 



1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

The Biology of Early Organelles of the Secretory Pathway 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: R.D. Klausner, Chief, CBMB 

Others: J. Donaldson, Senior Staff Fellow, CBMB, NICHD 

F. Hochstenbach, Visiting Fellow, CBMB 

E. Rabinovich, Guest Researcher, CBMB 

H. Radhakrisha, IRTA, CBMB 



COOPERATING UNITS (if any) 

None 



LAB/BRANCH 



Cell Biology and Metabolism Branch 



SECTION 

Section on Organelle Receptor Structure and Function 



INSTITUTE AND LOCATION 

NICHD, Bethesda, MP 



TOTAL STAFF YEARS: 
4.25 



PROFESSIONAL: 

3.25 



OTHER: 
1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 

D (a1 ) Minors 
n (aP) Intfirvifiws 



D (b) Human tissues Kl (c) Neitiier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The central vacuolar system of eukaryotic cells is an interlocking membrane system composed of 
distinct organelles (including ER, Golgi apparatus, secretory vesicles, secretory granules, 
plasma membrane, endosomes and lysosomes) and selective transport pathways. Proper 
functioning of this system is vital for regulating surface expression and secretion of 
proteins. Using the fungal metabolite brefeldin A (BFA) , which dramatically alters both the 
distribution and flow of membrane through the central vacuolar system, this group has focused 
on how organelles maintain their structure and identity, and how membrane trafficking between 
organelles is regulated. These questions have been directed to three different areas: (1) 
Understanding the biochemical basis of BFA action . We have found that BFA appears to affect 
the Golgi apparatus by acting on specific membrane targets that regulate the assembly of 
cytosolic coat proteins onto the cytoplasmic face of this organelle. Altered interaction of 
the coats with the organelle leads to organelle disassembly, tubule foimiation and mixing of 
organelle components within a defined "homotypic" membrane system. The biochemistry of two 
BFA sensitive coat proteins, _-COP and ARF are under investigation. (2) The biology and 
biochemistry of ARF family proteins. The recognition that the activation of the family of 
small GTP binding proteins called ARF's is most likely the target for BFA has led us to an 
extensive study of the biochemistry and cell biology of individual ARF family proteins using 
in vitro reconstitution of biochemical assays, mutagenesis and expression of wild type and 
mutant ARF proteins in cells. (3) Characterization of the distribution and flow of membrane 
within the vacuolar system. We have found that BFA causes the various compartments of the 
vacuolar system to collapse into new steady states which produce isolated but functioning new 
organelle units. Traffic within but not between these units continues in the presence of BFA. 
In summary, our studies with BFA provide new insights about the properties of sets of 
organelle-specif ic coat proteins and presents a framework for relating the biochemical 
regulation of membrane transport to the structure and maintenance of organelles . We have 
continued studies on the mechanisms of retention of proteins within the early organelles of 
the vacuolar system. 



PHS 6040 (Rev. 5/92) 



u 



ZOl-HD-01606-07 
THE BIOLOGY OF EARLY ORGANELLES OF THE SECRETORY PATHWAY 

Objectives 

Over the past year our efforts have focused on the mechanisms regulating cell shape and membrane 
traffic at the plasma membrane. We have pursued three projects in this area: 

1) The role of the ARF6 GTPase in the control of membrane traffic and membrane modeling at 
the plasma membrane. 

2) Endocytosis and plasma membrane protein turnover in the budding yeast Saccharomyces 
cerevisiae. 

3) Cell shape and cell polarity in the fission yeast Schizosaccharomyces pombe. 

ARF6: A GTPase implicated in plasma membrane modeling, actin rearrangements and formation of 
pseudopodia. 

Over the past several years, we have been studying the ARF (ADP-ribosylation factor) family of GTP 
binding proteins and their role in membrane traffic. We had showed that the ARFl and ARF3 
proteins function to regulate transport along the secretory pathway through the assembly of cytosolic 
coat proteins with membranes of the Golgi apparatus. There are at least three other distinct ARF 
family members, ebiquitously expressed in all cells. To begin to define the function of the other ARF 
proteins, we have analyzed the localization and cellular phenotypes of transiently expressed, epitope- 
tagged ARF proteins and their mutants in mammalian cells. For ARF6, we found that in cells 
overexpressing the wild type protein, the ARF6 protein is associated with the plasma and endosomal 
membranes, and that cellular morphology was normal. A mutant ARF6 predicted to be defective in 
GTP binding (T27N), and hence predominantly in the GDP state, is associated exclusively with 
endosomes, whereas a GTP hydrolysis-defective mutant (Q67L) is confined to the plasma membrane. 
The morphology of cells expressing these mutant forms of ARF6 is altered: cells expressing T27N 
exhibit an accumulation of tubular endosomal structures and cells expressing the Q67L GTP-active 
mutant exhibit an elaboration of plasma membrane folds at peripheral edges of these cells. 

Li order to better define the ARF6 function, we tested various pharmacologic agents for the ability 
to shift the distribution of ARF6 and the phenotype of cells overexpressing the wild type protein into 
that of cells overexpressing the active, GTP-hydrolysis defective mutant (Q67L). We found that 
treatment of HeLa cells transfected with wild type, epitope-tagged ARF6 with the G protein 
activator, aluminum fluoride (AlF) resulted in a redistribution of both ARF6 and actin to discrete sites 
on the plasma membrane which became increasingly protrusive over time. These structures resemble 
pseudopodia in that they contain actin, membranous folds and are dynamic structures forming and 
disassembling over a period of minutes. Immuno-EM showed that these pseudopodia contained 
numerous membranous folds similar to those in cells expressing the GTP-hydrolysis mutant 
ARF6/Q67L. The effects of AIF were reversible and specific to cells transfected with wild type 



ZOl-HD-01606-07 

ARF6; pseudopodia were not observed in cells transfected with non-myristoylated ARF6/G2A, GTP- 
binding defective ARF6/T27N, wild type ARFl, or in untransfected cells. Thus, it appears that AlF 
treatment results in the reversible "activation" of ARF6, allowing us to better characterize the ARP6 
effector ftinction, the extreme of which is the Q67L- induced extensive membranous folds. The AlF- 
induced pseudopodia in ARF6-overexpressing cells were distinct from actio rearrangements observed 
in cells overexpressing the Rac 1 protein, a GTPase reported to be involved in membrane ruffling. 
These cells formed lamellopodia when treated with ALF, and mimicked the phenotype observed in 
cells expressing the Q61L, GTP active Racl mutant. 

In addition to actin recruitment and rearrangements, other actin-associated proteins including gelsolin, 
focal adhesion kinase (FAK), and cortactin, were also recruited into the pseudopodia. Coupled to 
the dynamic pseudopodia formation is an apparent increase in the amount of membrane turnover via 
fluid phase pinocytosis. During AlF-induced pseudopod formation, fluid phase markers were rapidly 
and transiently taken up into macropinosomes within the pseudopods. This striking localized 
turnover of plasma membrane however, did not appear to significantly alter either transferrin uptake 
into the cell nor the distribution of clathrin AP-2 complexes along the plasma membrane. These 
results suggest that ARF6, like the Rho-related GTPases, can regulate the actin cytoskeleton and 
induce changes in plasma membrane architecture. We are currently investigating the requirements 
for the AlF induced pseudopod formation and find that arachidonic acid metabolism, specifically 
leukotriene synthesis, is involved in this process. 

Surface Protein Turnover in Saccharomyces cerevisiae 

The research over the past year has addressed the means by which S. cerevisiae achieves copper 
homeostasis via regulation of turnover of its plasma membrane copper transporter Ctrl p. Since 
surface protein turnover is expected to be mediated by internalization and delivery to the vacuole 
where hydrolases reside, this system should provide a setting for the study of endocytosis and 
intracellular trafficking in yeast. 

The Ctrlp is a copper transporter in the plasma membrane of S. cerevisiae that provides the limiting 
step for high affinity copper uptake into the cell. Maintenance of copper homeostasis is thus likely 
to involve modulation of cell surface levels of Ctrlp. We have demonstrated that Ctrlp is a stable 
protein under conditions of low copper but that addition of high copper (micromolar levels) triggers 
degradation of cell surface Ctrlp. This degradation is seen whether Ctrlp synthesis and expression 
is controlled by its own copper-responsive promoter, or by a heterologous galactose-inducible 
promoter. 

We then addressed the mechanism by which copper-triggered Ctrlp degradation was occuring. 
Contrary to expectation, we find that internalization and delivery to the vacuole does not appear to 
be the prinicpal mechanism that is responsible for this degradation. Copper dependent degradation 
of Ctrlp takes place in a number of mutant strains defective in endocytosis and vacuolar hydrolysis. 
Our results suggest that we may have uncovered a novel pathway of surface proteolysis in yeast that 
regulates surface expression of proteins. 



H 



ZOl-HD-01606-07 

Further experiments under conditions of lowered temperature indicate, however, that endocytosis and 
delivery to the vacuole may play a minor role in copper-dependent degradation of Ctrl p. We have 
also found that brief exposure to copper induces a shift of Ctrlp into a Triton-insoluble fraction, 
implying that a change in the physical state of Ctrlp may be involved in copper-dependent 
degradation. 

Cell Shape and Cell Polarity in Fission Yeast 

We want to understand how cells control cell morphogenesis and cell polarity. Cells possess 
specialized shapes that accommodate their fiinction. One aspect of cell morphogenesis is cell polarity, 
the abihty of the cell to establish spatially distinct domains that fulfill distinct functions. This 
phenomenon is widespread in biology and can be identified in both prokaryotes and eukaryotes. We 
have chosen fission yeast Schizosaccharomyces pombe as a model organism to start studying cell 
shape and cell polarity because it is an unicellular eukaryote with a well-defined cylindrical rod shape. 
The length of the cell increases as the cell grows, and this cell growth is polarized; it only occurs at 
the tips of the cells and not at the cylindrical middle part. In other words, new cell wall material is 
incorporated specifically at the cell tips, probably involving the secretory pathway. The main focus 
of our study in Schiz. pombe is to understand how cells establish, maintain, and control polarized cell 
growth. 

Our strategy for identifying new proteins and pathways that regulate cell morphogenesis and polarity 
consists of the generation of a panel of cell shape mutants of Schiz. pombe that are round, in contrast 
to wild-type cells which are rod-shaped. Wild-type cells were treated with the mutagen ethyl methane 
sulfonate and rounded mutants were isolated visually. Those mutants with a single gene defect were 
analyzed further. 

1) Characterization of J3 cell shape mutant: 

In our first cloning attempt, we chose the recessive mutant J3 because its cell shape shows a 
remarkable temperature-dependence. At the standard growth temperature, the cells are round and 
pear-shaped. However, its cell shape phenotype changes upon changes of the growth temperature. 
At a lower temperature, J3 cells display a wild-type cylindrical rod-shape. This property allows us 
to induce the mutant phenotype through a simple temperature change. At a higher, nonpermissive 
temperature, the cells showed an increase in the amount of membranes of the secretory pathway over 
control cells, with an apparent abundance of endoplasmic reticulum and Golgi apparatus. Also, the 
septa and cell walls were several times thicker than those of control cells. Eventually, the mutant cells 
lyse at the nonpermissive temperature. 

2) Efforts to clone the complementing gene and multi-copy suppressor genes. 

We have started experiments to isolate the gene mutated in J3 cells. Because a standard gene rescue 
experiment did not allow identification of the mutant gene (see next paragraph), we are following a 
positional cloning approach. The J3 locus was found to map to a region on chromosome IE and one 



.15 



ZOl-HD-01606-07 

cosmid clone that covered this chromosomal region was found to complement the J3 temperature- 
sensitive phenotype. Experiments to subclone and sequence the complementing gene are in progress. 

In standard gene rescue experiments, a wild-type genomic library was introduced into the recessive 
mutant and transformants with a wild-type phenotype were selected. We isolated six distinct clones 
which represent extragenic suppressors for mutant J3. To determine the nature of these suppressor 
clones, we sequenced four of the six genomic clones. Interestingly, all four clones contained a partial 
open reading from (ORP). One clone showed sequence similarity to a Sacc. cerevisiae cell wall 
enzyme P-l,3-glucanase, a second showed sequence similarity to the Sacc. cerevisiae cell surface 
protein, while the two other clones bore no similarity to known proteins. All four ORFs were partial, 
because none of the genomic inserts contained a termination codon. When the protein sequences 
were analyzed for the presence of a shared motif that might be responsible for the multi-copy 
suppression, we observed an amino-terminal hydrophobic signal sequence in all four ORFs, indicating 
that these polypeptides enter the secretory pathway. 

The finding that overexpression of secretory polypeptides might correct the defect of the J3 mutant 
is significant, considering the observation that the mutation causes changes in the secretory pathway, 
that, in turn, might be responsible for the changes in cell shape. 



.16 



ZOl-HD-01606-07 

Publications 



Donaldson JG, Radhakrishna H, and Peters PJ. The ARF GTPases: Defining roles in membrane 
traffic and organelle structure. Cold Spring Harbor Symposium on Quantitative Biology, Vol 60, 
1995(In press). 



Peters PJ, Hsu VW, Ooi CE, Finazzi D, Teal SB, Oorschot V, Donaldson JG and Klausner RD. 
Overexpression of wild-type and mutant ARFl and ARF6: Distinct perturbations of nonoverlapping 
membrane compartments. J Cell Biol 1995;128:1003-1017. 



17 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl-HD-01607-05 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT 180 characters or less. Title must fit on one line between the borders.) 

Protein Trafficking in the Secretory Pathway 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: J. Bonifacino, Visiting Scientist, CBMB 
Others :H. Bosshart, Guest Researcher, CBMB 

M. Delahunty, IRTA, CBMB 

M.C. Fournier, Visiting Fellow, CBMB 

M.S. Marks, IRTA, CBMB 

H. Ohno, Visiting Fellow, CBMB 

P. Voorhees, HHMI-NIH Scholar, CBMB 



COOPERATING UNITS (if any) 

P.Peters, University of Utrecht, The Netherlands; T. Kirchhausen, Harvard 
University. 



LAB/BRANCH 

Cell Biology and Metabolism Branch 



SECTION 

Unit on Intracellular Protein Trafficking, Section on Organelle Receptor & Function 



INSTITUTE AND LOCATION 

NICHD, Bethesda, MD 



TOTAL STAFF YEARS: 
6.8 



PROFESSIONAL: 
5.0 



OTHER: 
1.8 



CHECK APPROPRIATE B0X(ES1 

D (a) Human subjects D (b) Human tissues 
D (a1) Minors 
D (a2) Interviews 



a (c) Neither 



SUMMARY OF WORK lUse standard unreduced type. Do not exceed the space provided.) 



This group investigates the molecular mechanisms that determine the intracellular localization 
and sorting of integral membrane proteins within the endocytic and secretory pathways. This 
past year, our work has been directed towards identifying and characterizing: (i) structural signals 
that specify localization and sorting of integral membrane proteins, (ii) proteins that recognize 
the sorting signals, and (iii) other components of the sorting machinery. 

The nature of signals involved in sorting of integral membrane proteins was investigated using as 
models a molecule involved in antigen presentation, HLA-DM, and the endopeptidase, furin. 
HLA-DM is localized to an endosomal/lysosomal compartment where class II molecules of the 
major histocompatibility complex (MHC) encounter and bind antigenic peptides. Our studies 
showed that targeting of HLA-DM to this compartment is largely mediated by a tyrosine-based 
signal (YTPL) in the cytoplasmic domain of the /3 chain of the molecule. Furin is predominantly 
localized to the trans-Goi^i network (TGN). Molecular dissection of furin demonstrated the 
existence of two targeting signals that contribute to the localization of furin to the TGN: 
tyrosine-based signal (YKGL) and a novel acidic sequence (WQEECPSDSEEDEGRGER). This 
novel acidic signal was shown to be a site of phosphorylation by casein kinase II, suggesting a 
role for phosphorylation in regulating the trafficking of furin. 

A search for proteins that bind to tyrosine-based signals resulted in the identification of the 
medium chains (/Xj and y.^ of clathrin-associated adaptor complexes (AP-1 and AP-2) as the 
signal-recognition components of the sorting machinery. This finding opened the way for a 
detailed characterization of the interaction of signals and recognition proteins at a molecular 
level. 



PHS 6040 (Rev. 5/92) 



ZOl-HD-01607-05 
PROTEIN TRAFFICKING IN THE SECRETORY PATHWAY 

Project Description 

Objectives: 

This group investigates the molecular mechanisms that determine the localization and 
sorting of integral membrane proteins within the endocytic and secretory pathways. The 
main objectives of this project are the identification of signals involved in protein 
localization and sorting within the trans-Go\gi network (TGN) and the 
endosomal/lysosomal system and of recognition molecules that bind to the signals. 

Methods Employed 

All projects in this group involved the use of recombinant DNA procedures, morphologic 
and biochemical techniques, and yeast genetic approaches. In order to search for sorting 
signals within several integral membrane proteins, we constructed mutant and chimeric 
proteins using the polymerase chain reaction (PCR) and other standard recombinant DNA 
procedures. Constructs were expressed in mammalian cells by either transient or stable 
transfection. The subcellular localization of the different protein constructs was determined 
by immunofluorescence microscopy, image analysis, and quantitative immunoelectron 
microscopy, and their biosynthesis, processing and degradation were studied by pulse-chase 
metabolic labeling and immunoprecipitation. The ability of signals to mediate 
internalization from the cell surface was examined using quantitative endocytosis assays. 
We also examined the phosphorylation and dephosphorylation of some of the sorting 
signals by labeling with ^^P-phosphate both in vivo and in vitro. Proteins that bind to 
sorting signals (recognition molecules) were identified using a yeast two-hybrid system and 
affinity purification with glutathione-S-transferase (GST) fusion proteins. The interaction of 
sorting signals with their recognition molecules was characterized using various in vitro 
binding assays and surface plasmon resonance spectroscopy. Complementary DNAs 
encoding the recognition molecules and related proteins were cloned using established 
recombinant DNA procedures. 

Major Finding s 

Cytoplasmic Sorting Signals. 

It is now well established that the localization of integral membrane proteins to specific 
compartments of the secretory pathway is mediated by information encoded within the 
structure of the proteins. In some cases, this information derives from a global 
physicochemical property of the proteins, such as a tendency to aggregate in certain cellular 
environments. In other cases, information is contained within short, linear arrays of amino 
acid residues that act as specific sorting ("address") signals. Our group has been particularly 
interested in the mechanisms that determine protein localization to compartments of the 



.19 



ZOl-HD-01607-05 

peripheral secretory pathway, such as the TGN, lysosomes and endosomes, for which 
cytoplasmic sorting signals play a major role. The sorting signals are thought to interact 
with cytoplasmic receptor-like molecules that function as recognition components of the 
sorting machinery. The main objectives of our work this year have been the identification 
of sorting signals within proteins that are targeted to the TGN and the 
endosomal/lysosomal system and the search for the specific signal-recognition molecules. 

A Tyrosine-based Signal Targets a Component of the Antigen Presentation Machinery, 
HLA-DM, to the Endosomal/Lysosomal System. 

HLA-DM is a membrane-bound heterodimer (q!/3) that promotes loading of antigenic 
peptides onto class II molecules of the major histocompatibility complex (MHC). In 
antigen presenting cells, HLA-DM is localized to a compartment of the 
endosomal/lysosomal system known as the MIIC (for MHC class 11 compartment). 
We conducted a systematic analysis of sorting information within HLA-DM and found that 
the localization of HLA-DM to the MIIC was directed by a tyrosine-based signal (YTPL) 
within the cytoplasmic domain of the jS chain of the molecule. This signal was active both 
as an internalization and lysosomal targeting signal, thus establishing a biogenetic 
relationship of the MIIC with the endosomal/lysosomal system. 

Two Independent Targeting Signals Mediate Localization of the Endopeptidase. Furin, to 
the TGN. 

Turin is a dibasic endopeptidase responsible for the proteolytic processing of numerous 
protein precursors, including pro-hormones and viral envelope glycoprotein precursors. 
Our previous studies had demonstrated that furin was predominantly localized to the TGN 
and that the cytoplasmic domain was both necessary and sufficient for TGN localization. 
This past year, we have performed a systematic analysis of targeting information within the 
furin molecule. Our studies have uncovered the existence of two targeting signals that 
contribute to the TGN localization of furin. The first signal is a typical tyrosine-based 
motif, YKGL. The second signal is distinct from other signals that have been described to 
date. It consists of a strongly hydrophilic sequence containing a cluster of acidic amino acid 
residues (WQEECPSDSEEDEGRGER). Both signals can mediate internalization from the 
cell surface, but only the acidic sequence is capable of conferring localization to the TGN 
per se. We also found that two serine residues within the acidic sequence are 
phosphorylated by casein kinase II in vivo and in vitro. This observation suggests a role for 
phosphorylation in regulating the localization and trafficking of furin. 

Identification of the Medium Chains of Clathrin-associated Adaptor Complexes as the 
Recognition Molecules for Tyrosine-based Sorting Signals. 

During the past year, we undertook several approaches to identify molecules that bind to 
tyrosine-based signals and thereby mediate their sorting functions. Using a yeast two-hybrid 
system, we found that the medium (^) chains of clathrin-associated adaptor complexes 



20 



ZOl-HD-01607-05 

interact specifically with tyrosine-based signals. Both /ij (a component of TGN adaptors) 
and /X2 (a component of plasma membrane adaptors) are capable of binding to various 
tyrosine based signals, such as those of the proteins TGN38 (YQRL), HLA-DM (YTPL), 
LAMP-1 (YQTI), CD68 (YQPL) and the transferrin receptor (YTRF). In vitro binding 
assays confirmed the results of the two-hybrid system and allowed a detailed 
characterization of the interactions. The identification of the jx-^ and /X2 as the recognition 
molecules for tyrosine-based signals supports a role of clathrin coats and tyrosine-based 
signals in sorting at both the TGN and the plasma membrane. 

In Vivo Studies of the Saturability of Sorting Processes Mediated by Tyrosine-Based Signals. 

The existence of specific recognition molecules that bind to tyrosine-based sorting signals 
predicts that processes mediated by such signals should be saturable. We tested this 
prediction by overexpressing in transfected cells proteins that have tyrosine-based signals 
and analyzing the surface expression of other proteins that are normally targeted to 
lysosomes by virtue of tyrosine-based signals. We observed that such overexpression 
interfered with several processes dependent on tyrosine-based signals such as lysosomal 
targeting and internalization from the cell surface. These studies were extended to the HIV- 
1 envelope glycoprotein complex, which also has a tyrosine-based signal that is active in 
endocytosis. Overexpression of the envelope glycoprotein resulted in missorting of 
lysosomal proteins to the cell surface, a phenomenon that may explain some of the 
cytopathic effects of HIV-1 on the host cells. 

Proposed Course of Research 

The identification of the medium adaptor chains as the receptors for tyrosine-based signals 
has had a major impact in our ability to study the molecular mechanisms of protein 
sorting. We now plan to undertake a detailed characterization of the signal-receptor 
interactions, making use of various binding assays developed over the past year. A major 
priority will be to establish the structural features of the signals that determine the 
specificity and avidity of their binding to the receptor molecules. Other factors that may 
affect their sorting function such as their placement within the cytoplasmic domain and 
their multiplicity will also be investigated. We also plan to conduct structure-function 
analyses of the medium chains, for the purpose of identifying the signal-binding domains, 
sites of interaction with other adaptor chains, regulatory regions, etc. This information on 
the medium chains will be used to construct dominant mutants that could be used to study 
the role of these chains in various sorting processes in intact cells. Since ^1 and ^2 ^I's 
members of a growing superfamily of related molecules, we will investigate whether the 
other members also have a signal recognition function. If this is the case, we will determine 
whether the medium chain homologs are components of novel adaptor complexes and 
what their subcellular localizations and functions are. We will also attempt the 
identification of proteins that bind to the acidic signal in furin using techniques similar to 
those that led to the identification of the medium adaptor chains as the receptors for 
tyrosine-based signals. 



21 



ZOl-HD-01607-05 



Publications. 

Delahunty M and Bonifacino J. Disorders of intracellular protein trafficking in human 
disease. Conn Tissue Res 1995 (In press). 

Marks MS, Germain RN, and Bonifacino JS. Transient aggregation of major 
histocompatibility complex class II chains during assembly in normal spleen cells. J Biol 
Chem 1995;270:10475-81. 

Marks MS, Roche P, van Donselaar E, Woodruff L, Peters PJ, and Bonifacino JS. A 
lysosomal targeting signal in the cytoplasmic tail of the (3 chain directs HLA-DM to the 
MHC class II antigen processing compartment. J Cell Biol 1995(In press). 

Ohno H, Stewart J, Fournier MC, Bosshart H, Rhee I, Miyatake S, Saito T, Gallusser A, 
Kirchhausen T and Bonifacino JS. Interaction of tyrosine-based sorting signals with 
clathrin-associated proteins. Science(In press). 

Rajasekaran AK, Humphrey JS, Wagner M, Miesenbock G, Le Bivic A, Bonifacino JS, 
Rodriguez-Boulan E. TGN38 recycles basolaterally in polarized MDCK cells. Mol Biol Cell 
1994;5:1093-103. 

Voorhees P, Deignan E, van Donselaar E, Humphrey J, Marks MS, Peters PJ and 
Bonifacino JS. An acidic sequence within the cytoplasmic domain of furin functions as a 
determinant of TGN localization and internalization from the cell surface. EMBO J(In 
press). 



4t.. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl-HD-01608-5 



PERIOD COVERED 

October 1, 1994 to September 30, 



1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Gene Regulation in Response to Environmental Stress 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
PI: G. Storz, Senior Staff Fellow, CBMB 
Others :E. Bruggemann, IRTA, CBMB 

C. Essex, Pre-IRTA, CBMB 

C. Merlotti, Visiting Fellow, CBMB 

A. Zhang, IRTA, CBMB 



COOPERATING UNITS (if any) 

None 



LAB/BRANCH 



Cell Biology and Metabolism Branch 



SECTION 



Unit on Environmental Gene Regulation 



INSTITUTE AND LOCATION 

NICHD, Bethesda, MP 



TOTAL STAFF YEARS: 

4 



PROFESSIONAL: 
4 



OTHER: 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 

n (a1) Minors 
n (aP) IntRrvJRWs 



n (b) Human tissues (c) Neitiner 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This group studies how organisms sense environmental signals and transduce the signals into changes in gene 
expression and cell physiology. The group has continued their characterization of the two regulators centra! 
to the Escherichia coli response to oxidative stress: OxyR, a 34 kDa transcriptional activator which i* 
switched on by oxidation, and OxyS, a novel 109 nucleotide RNA regulator which acts as an activator anc 
repressor of gene expression. In addition to studying the mechanisms of OxyR and OxyS action, the group 
has begun to determine what known regulators are important to the response to hydrogen peroxide ir 
Saccharoinyces cerevisiae. Using Arabidopsis thaliana as a model system, the group is also characterizing 
mutants defective in their developmental response to blue light. 



n 



PHS 6040 (Rev. 5/92) 



ZOl-HD-01608-05 
GENE REGULATION IN RESPONSE TO ENVIRONMENTAL STRESS 

Project Description 

Objectives: 

This project is aimed at understanding the molecular mechanisms whereby organisms perceive an 
environmental signal and transduce this information into a change in gene expression. In one 
subproject, we are characterizing the E. coli and S. cerevisiae responses to oxidative stress, and in 
a second subproject, we are characterizing Arabidopsis mutants to elucidate how plant 
development is regulated by light. 

Methods Employed: 

Recombinant DNA techniques employed include the isolation of RNA and DNA, preparation of 
recombinant plasmids, molecular cloning, nucleic acid electrophoresis, DNA sequencing, 
Northem and Southern blotting, chemical synthesis of deoxyoligonucleotides, DNA amplification 
via the polymerase chain reaction, cDNA Ubrary construction and subtraction. Biochemical 
approaches used include fast protein Uquid chromatography (FPLC) for the protein purification, 
DNA binding and in vitro transcription assays, and immunoblotting. Standard genetic crosses and 
the generation of lacZ marker gene fusions by transposition or by recombination onto Lambda 
followed by integration are also being carried out. 

Oxidative Stress Responses in Bacteria and Yeast. 

Reactive oxygen species (Oj* , ^ Q , and HO») can lead to the damage of almost all cell 
components (DNA, Upid membranes, and proteins) and have been implicated as causative agents 
in several degenerative diseases. Most organisms have an adaptive response to defend against 
oxidants. Treatment of both bacterial and yeast cells with low doses of H2O2 results in the 
induction of a distinct group of proteins, the decreased expression of other proteins and resistance 
to killing by subsequent higher doses of HjOj. The bacterial response to P P involves at least 
two regulators. The expression of nine of the H202-inducible proteins is controlled by the OxyR 
protein which is homologous to the "LysR" family of bacterial regulators. The oxidized but not 
the reduced form of the OxyR protein activates transcription, suggesting that oxidation of OxyR 
brings about a conformational change that leads to RNA polymerase activation. Treatment with 
hydrogen peroxide also leads to the induction of a unique RNA regulator denoted OxyS. 
Previous studies by this group have shown that OxyS is 109 nucleotides in length and is not 
translated, and several target genes that are activated or repressed by the OxyS RNA have been 
identified. Less is known about the regulation of the yeast defenses against hydrogen peroxide, 
however, regulators of metal homeostasis may also be critical to the oxidative stress response. 

Characterization of OxyR domains. We have characterized six different non-binding and nine 
constimtively-active mutants of OxyR. Five of the mutations causing the DNA-binding defect 



24 



ZOl-HD-01608-05 

map near the N-terminal helix-tum-helix motif conserved among the LysR family members, 
confirming that this region is a DNA-binding domain in OxyR. The sixth non-binding mutant 
(E225K) was found to be predominantly dimeric in contrast to the tetrameric wild type protein, 
suggesting that a C-terminal domain defined by the E225K mutation is involved in 
multimerization. The mutations causing the constitutive phenotype are located in the carboxy- 
terminal two thirds of the protein and five of the mutations map near a cysteine residue (CI 99) 
critical for the redox-sensitivity of OxyR. In vivo as well as in vitro transcription experiments 
showed that the constitutive mutant proteins were able to activate transcription under both 
oxidizing and reducing conditions, and DNase I footprints showed that the activation is due to the 
ability of the mutants to induce cooperative binding of RNA polymerase. The mutant proteins are 
now useful tools for the biochemical characterization of the redox-active center in OxyR. 

Regulation by the OxyS RNA. We have also continued our studies of the novel, 109 nucleotide 
OxyS RNA. We previously found that OxyS acts in trans to repress the expression of fhlA 
(encoding an activator of anaerobic genes), dps (encoding a non-specific DNA binding protein), 
acrH (encoding a metal resistance/nodulation/cell division-type transporter), yhiM (a gene with 
homology to GABA transporters), and gadB (encoding glutamate decarboxylase). OxyS also 
activates expression of uhpT (encoding a hexose phosphate transporter) and pqqE (required to 
synthesize the co-factor PQQ). We have now subcloned regions of the fhlA, dps, yhiM, and gadB 
promoters in order to define the OxyS response elements. Interestingly, while OxyS represses the 
transcription of dps, yhiM, and gadB, the RNA acts to repress the translation oifhlA. Additional 
studies of the OxyS RNA have shown the the half-life of the RNA is greater than 20 minutes and 
only the 3' 46 nucleotides are essential for function. Cross-species hybridization also revealed that 
while the OxyS RNA is present in closely-related E. coli. Salmonella, and Shigella, it is not 
detected in Klebsiella, Pseudomonas, or Enterobacter. 

Roles of Regulators of Metal Homeostasis in the Yeast Response to Oxidative Stress. Relatively 
little is known about the cellular mechanisms used by yeast to protect against oxidative damage. 
Since metals contribute to the production of oxygen radicals, we are investigating the effects of 
known regulators of metal homeostasis on the S. cerevisiae response to oxidative stress. We 
constructed congenic haploid strains carrying single and double knockouts of genes encoding 
regulators of metal homeostasis (AFTl, MACl, ACEl) and regulators conferring pleiotropic 
metal and drug resistance {YAPl, CADI). The growth rates and the oxidant sensitivities of these 
strains are now being determined in a variety of growth assays. This approach has already shown 
that YAPl, AFTl, and MACl play a role in protecting the yeast cells against oxidative stress and 
wUl provide a foundation for the identification of additional components of the yeast defense 
response. 

Blue Light Regulation in Arabidopsis. 

The normal development of plants is generally dependent on light. Spectral studies have shown 
that plants undergo morphological changes in response red/far red, blue, and UV light. For 
example, dark grown plants have extremely elongated hypocotyls (stems) compared to light- 



21) 



ZOl-HD-01608-05 

grown seedlings. At present, the mechanisms by which the Ught signals are transduced into 
changes in plant growth and morphology are largely unknown. 

Characterization of hy4 and hy5 mutants. We took advantage of the difference in hypocotyl 
length to screen for Arabidopsis mutants that have elongated hypocotyls under blue light. These 
mutants are impaired either in their ability to sense hght of this wavelength or in their ability to 
transduce the Ught signal into the appropriate developmental response. 24 independent mutants 
defective in their developmental response to blue Ught were isolated from a screen of 300,000 
mutagenized seeds. The mutants feU into two complementation groups corresponding to the HY4 
and HY5 genes. Since HY4 is thought to encode the blue light photoreceptor, 14 hy4 mutants are 
now being characterized to determine the nature of the mutations. These studies are being 
complemented by subtractive screens between wild type, hy4 and hy5 cDNA Ubraries to identify 
genes that are differentially expressed between the wild type and mutant seedlings. 

Characterization of tt mutants. One response of Arabidopsis seedlings to blue Ught is elevated 
production of purple anthocyanin pigments. Since mutants that do not produce the pigments are 
easily detected, eleven pigment-defective mutants {ttl-ttlO, ttg) have been isolated. In a 
collaboration with Drs. B. Shirley (Virginia Polytechnic Institute and State University), H. 
Goodman, W. Kubasek, F. Ausubel (Massachusetts General Hospital) and M. Koomneef 
(Agricultural University, Wageningen), we have helped to characterize these tt mutants to 
determine the nature of the defects. 



PG 



ZOl-HD-01608-05 



Publications: 

KuUik I, Toledano MB, Tartaglia LA, Storz G. Mutational analysis of the redox-sensitive 
transcriptional regulator OxyR: Regions important for oxidation and transcriptional activation. J 
Bacteriol 1995;177:1275-84. 

KuUik I, Stevens J, Toledano MB, Storz G. Mutational analysis of the redox-sensitive 
transcriptional regulator OxyR: Regions important for DNA-binding and multimerization. J 
Bacteriol 1995;177:1285-91. 

Shirley BW, Kubasek WL, Storz G, Bruggemann E, Koomneef M, Ausubel FM, Goodman HM. 
Analysis of Arabidopsis mutants deficient in flavonoid biosynthesis. Plant J 1995;(In press). 



27 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl-HD-01609-04 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 
Localization and Dynamics of Intracellular Organelles 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
PI: J. Lippincott-Schwartz, Senior Staff Fellow, CBMB, NICHD 

N. Cole, Pre-IRTA, CBMB, NICHD 

J. Ellenberg, Special Volunteer, CBMB, NICHD 

N. Sciaky, Special Volunteer, CBMB, NICHD 

J. Song, Pre-IRTA, CBMB, NICHD 



COOPERATING UNITS (if any) 

Mark Terasaki, University of Connecticut; Michael Edidin, Johns Hopkins University; 
Eric Siggia, Cornell University; Carolyn Smith, NIMH, LCM, NIH. 



LAB/BRANCH 

Cell Biology and Metabolism Branch 



SECTION 

Unit on Organelle Biology 



INSTITUTE AND LOCATION 

NICHD, Bethesda, MD 



TOTAL STAFF YEARS: 
5.0 



PROFESSIONAL: 
3.0 



OTHER: 
2.0 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 

D (a1) Minors 
n (aP) IntRrvJRWS 



n (b) Human tissues Kl (c) Neitlier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

How membrane-bound organelles of eukaryotic cells maintain their identity and subcellular localization amidst an enormous input and outflow 
of membrane and protein is a central question in cell biology. Studies in this group have focused on this question and have sought to define the 
cellular and molecular mechanisms which underlie the organization and distribution of eukaryotic organelles. Particular attention has been paid 
to the Golgi complex which plays a fundamental role in the processing and sorting of protein moving through the secretory pathway. The Golg 
complex in higher eukaryotes consists of stacks of flattened cistemae usually localized to the perinuclear region near the microtubular 
organizing center (MTOC). Recent studies have suggested this organization and positioning of the Golgi are controlled by dynamic processes. 
Tubulovesicular structures emerging from Golgi elements along microtubules, for example, enable adjacent Golgi stacks to communicate. In 
addition, reversible dispersal of Golgi elements occurs during microtubule disruption, mitosis and brefeldin A (BFA)-treatment. To further 
understand these processes and their relationship to the three-dimensional morphology and function of the Golgi we have taken four major 
approaches. 1) Imaging of the Golgi complex in living cells . Using the vital dye BODIPY-ceramide to label the Golgi complex in living cells 
we have performed time-lapse imaging to examine the dynamics of the Golgi complex in normal and BFA-treated cells. The important role of 
membrane tubulation in the normal dynamics and maintenance of Golgi structure within cells is under investigation. 2) Role of microtubules in 
Golgi localization and traffic . We have found that microtubules are important for both localizing the Golgi complex to the MTOC and for 
facilitating anterograde (ER-to-Golgi ) and retrograde (Golgi-to-ER) membrane traffic into and out of this central region. Our characterization 
of Golgi dispersal induced by microtubule-disruption has suggested a mechanism involving membrane transport of Golgi components along 
retrograde and anterograde membrane pathways rather than diffusion or actin/myosin based transport. The role of microtubules in facilitating 
and directing membrane traffic to and from the Golgi, therefore, is likely to underlie the subcellular location of this organelle. 3) Role of 
microtubule motors in Golgi traffic . We have found that the microtubule motor protein, kinesin, associates with the anterograde and retrograde 
pathways leading to and from the Golgi complex. Conditions of microtubule disruption and low temperauire treatments, which slow retrograde 
traffic, result in a large accumulation of kinesin on Golgi membranes. Factors that might regulate kinesin motor activity as this molecule cycles 
through anterograde and retrograde pathways are under investigation. 4) Role of Golgi positioning in late processing events of the secretory 
pathway. We have found that microtubule-dependent positioning of the Golgi complex to the MTOC is likely to facilitate the interaction of 
components moving through secretory and endocytic pathways . 



PHS 6040 (Rev. 5/92) 



ZOl-HD-01609-04 
LOCALIZATION AND DYNAMICS OF INTRACELLULAR ORGANELLES 

Objectives: 

The unit on Organelle Biology at CBMB studies the ntiechanisms regulating the organization and 
distribution of organelles and transport intermediates comprising the endomembrane system of 
higher eukaryotes. Over the past year our group has focused on understanding the dynamics of 
membrane proteins comprising the Golgi complex, an organelle which plays a central role in the 
transport, processing and sorting of secretory products leaving the ER. We have pursued three 
major projects: (1) construction of Golgi protein chimeras containing the Aequorea victoria green 
fluorescence protein (GFP) to examine by fluorescence microscopy the mobility and diffusion 
rates of Golgi membrane proteins in living cells; (2) development of an assay system to study the 
extent and role of retrograde (Golgi-to-ER) transport by Golgi membrane proteins; and (3) 
elucidation of the mechanism(s) underlying Golgi dispersal. 

Golgi protein-GFP chimeras 

To gain insight into the dynamics and mobility of different proteins comprising Golgi membranes, 
we constructed Golgi protein chimeras containing GFP and imaged these proteins in living cells 
using fluorescence microscopy. GFP was attached to (1) the lumenal tail of galactosyltransferase, 
a Golgi resident enzyme (GFP-galtf); (2) the cytoplasmic tail of the BCDEL receptor, which resides 
at steady-state within the Golgi, but recycles to the ER in response to occupancy by KDEL- 
containing ligands (GFP-KDELR); and (3) the cytoplasmic tail of a putative recychng deficient 
KDEL receptor (GFP-KDELRm). In transiently transfected cells all of the GFP-chimeras were 
locahzed primarily to the Golgi complex. Overexpression of lysozyme-KDEL within cells caused 
GFP-KDELR, but not GFP-KDELRm, to redistribute into the ER, consistent with these two 
chimeras functioning respectively, as wildtype and defective KDEL receptors. Time-lapse 
recordings by confocal microscopy following the dynamics of each of the fluorescent chimeras 
revealed their distribution to include dynamic tubule processes which extended out and retracted, 
or disconnected from stable perinuclear Golgi elements. Detached, mobile tubule elements 
containing the GFP chimeras moved away from the Golgi along curvilinear microtubule tracks at 
rates of 0.6 um/sec before changing shape and direction, or fusing with other peripheral 
structures. 

Insight into the potential role of the tubule processes containing the GFP-Golgi protein chimeras 
came from experiments following their dynamics in cells treated with brefeldin A (BFA), a drug 
which redistributes all Golgi proteins into the ER. Minutes after adding BFA to cells Golgi tubule 
elements containing the chimeras became more numerous. These tubules resembled Golgi tubules 
in untreated cells, moving along microtubule tracks at 0.6 um/sec to the cell periphery. Unlike 
Golgi tubules in untreated cells, the BFA tubules failed to detach from central Golgi structures 
and within 3-4 min transformed the entire Golgi complex into a tubule network. Soon thereafter, 
fusion of ER and Golgi membranes occurred resulting in Golgi membranes emptying into the ER 
within 30 sec. These results raise the possibihty that Golgi tubule elements observed in control 



2d 



ZOl-HD-01609-04 

cells normally serve as intermediates in Golgi-to-ER retrograde traffic. In untreated cells, such 
tubule traffic would be highly regulated and involve a mechanism for detachment of retrograde 
tubules from Golgi cistemae. In BFA-treated cells, such traffic would no longer be regulated, and 
proliferation of retrograde tubules would result in the inability of the Golgi complex to maintain 
its distinct identity from the ER. Our ability to image retrograde traffic of Golgi membrane 
proteins in living cells induced either by BFA or by overexpression of KDEL ligand should prove 
useful in verifying this proposal. 

GFP-Golgi protein FRAP studies 

Currently, there is great interest in defining the mechanism(s) underlying the "retention" of Golgi 
membrane proteins in the Golgi complex. Two alternative models have been suggested: one 
involving immobilization by self interactions, or kin recognition between different Golgi proteins; 
and the other by lateral partitioning of Golgi proteins into specific lipid domains. To distinguish 
between these models we investigated the diffusional mobility of the GFP-chimeras within Golgi 
membranes using photobleach recovery techniques. Regions of elongated Golgi elements 
expressing each of the constructs were bleached using a confocal microscope and fluorescence 
recovery within this region observed by time-lapse fluorescence microscopy. Rapid recovery into 
the bleached zone was observed for all of the chimeras, indicating lateral diffusion of these 
proteins across Golgi cistemae. The diffusion rates were quantified using a laser microscope 
designed for fluorescence photobleaching recovery (FPR) measurements. A stripe of two microns 
wide was bleached across Golgi membranes. Within this geometry, fluorescence recovered to 
about 70% of pre-bleach levels. D from the rapid recovery of fluorecence was 1.2 XIO'^ cmVsec 
for GFP-KDELR, 1.1 X 10"^ cnf/sec for KDELRm, and 1.65 X'^O ^cm /sec for GFP-galtf 
These values are approximately that expected for unhindered lateral diffusion of a multispan 
protein in a membrane and are similiar to that of rhodopsin, in vertebrate visual membranes. Our 
estimates of D were 3-4 fold higher than that previously measured for VS V G protein labeled with 
a fluorescent antibody fragment while residing within the Golgi. AlF treatment did not affect D, 
but reduced the recovery of fluorescence to 30-40% of pre-bleach levels. This result is consistent 
with AlF's effect, which is to fragment Golgi membranes into vesicles smaller in diameter than the 
bleached line. Rapid mobility of the GFP-Golgi protein chimeras in Golgi membranes suggests 
that these proteins normally do not interact with components which retard their diffusion. 

Significantly, FPR measurements of GFP-galtf redistributed into the ER by BFA, revealed its 
diffusional mobility to be three times slower than that measured in Golgi membranes. GFP-galtf 
has a27 kD ectodomain, while GFP-KDELR has essentially none. Since the diffusional mobility of 
GFP-KDELR was the same whether it was in Golgi or in ER membranes (as a result of BFA 
treatment), it is possible that interactions between the ectodomain of a membrane protein and 
lumenal proteins comprising the ER can alter the diffusional mobility of the membrane protein. 
Consistent with this possibility, we found that when GFP-KDELR was redistributed into the ER 
by overexpression of KDEL ligand (which behaves as a soluble ER resident protein), the 
diffiisional mobility of KDELR in the ER was two fold slower than its mobility either in Golgi 
membranes or in ER membranes in the absence of overexpressed ligand. 



30 



ZOl-HD-01609-04 

Retrograde transport of Golgi membrane proteins 

Proper functioning of the secretory pathway in higher eukaryotes requires a significant retrograde 
flow of membrane and protein from the Golgi complex back to the endoplasmic reticulum (ER). 
This ensures return of escaped ER resident proteins, as well as membrane components necessary 
for continued anterograde traffic. A variety of molecules, including ERGIC-53, p58, and 
KDELR, have been shown to cycle constitutively between the ER and Golgi complex, although 
the exact routes these molecules travel is unclear. Whether Golgi resident proteins cycle through 
these pathways, as well, is unknown. To address this question we constructed chimeric Golgi 
proteins containing the luminal domain of the temperature sensitive viral glycoprotein, ts045VSV 
G, which misfolds and is retained within the ER at its restrictive temperature of 40°C, but at 32°C 
folds correctly and moves through the Golgi complex to the plasma membrane. We reasoned that 
if Golgi-targeted VSV G chimeras were to cycle between the ER and Golgi complex, then 
shifting the temperature to 40°C should result in their misfolding and retention in the ER as they 
cycled through this compartment. In transiently transfected cells at 32°C, VSVG chimeras 
(including VSV G attached to the transmembrane and cytoplasmic domains of TGN 38, KDELR 
and KDELRm) were found localized to the Golgi complex. Upon shiftmg the temperature to 
40°C, these proteins redistributed into the ER, without altering the distribution of other Golgi 
membrane markers. The kinetics of this process varied with each chimera, with complete 
redistribution occurring in as little as fifteen minutes, or as long as three hours. This process was 
fully reversible, as all chimeras showed a Golgi staining pattern within one hour of shift back to 
the permissive temperature. These observations suggest that Golgi membrane proteins cycle at 
some finite rate through pathways connecting the ER and Golgi complex. We are currently using 
the VSV G-Golgi chimeras to develop an assay system for retrograde transport in order to define 
the biocemical requirements of this transport pathway. 

Mechanismfs^ of Golgi dispersal 

Despite its distinctive morphology and localization within cells, the Golgi complex is capable of 
rapid disassembly and reassembly during mitosis, as well as under pharmacologically-induced 
conditions (e.g. BFA, ililumiquinone or okadaic acid), and reversibly redistributes to numerous 
peripheral sites in response to microtubule depolymerization. How Golgi fragmentation and 
dispersal during these processes is accomplished is far from being understood. To begin to 
understand the mechanisms underlying Golgi dispersal we analyzed this process in cells whose 
microtubules were depolymerizated. It has been know for years that microtubule disruption has 
dramatic effects on Golgi morphology, with hundreds of functional Golgi islands appearing 
throughout the cytoplasm. Surprisingly, membrane transport from the endoplasmic reticulum 
(ER) to the Golgi complex and subsequently to the cell surface is generally not affected. We 
analyzed this dispersal process using quantitative fluorescence microscopy and digital image 
processing. Golgi membrane components were found to redistribute to a distinct number of 
peripheral sites that were not randomly distributed, but corresponded to sites of protein exit from 
the ER. Whereas Golgi enzymes redistributed gradually over several hours to these peripheral 
sites, ERGIC-53 (a protein which constitutively cycles between the ER and Golgi) redistributed 
rapidly (within 15 minutes) to these sites after fu-st moving through the ER. Interestingly, 



81 



ZOl-HD-01609-04 

processing by Golgi enzymes of proteins moving through the secretory pathway was initially 
blocked after microtubule disruption. Once Golgi components had redistributed to peripheral 
sites, however, processing was as efficient as in untreated cells. This suggests that Golgi 
dispersal upon microtubule disruption is required for reestablishing secretory flow from the ER to 
the Golgi complex. Experiments examining the effects of microtubule disruption on the 
membrane transport pathways connecting the ER and Golgi suggested a potential role for these 
pathways in the dispersal process. Anterograde clustering of peripheral pre-Golgi elements into 
the central Golgi region was blocked by microtubule disruption, whereas retrograde traffic from 
the Golgi to the ER was only slighdy inhibited. Golgi dispersal, therefore, could result from a 
slow but constitutive flux of Golgi resident proteins through ER/Golgi cycling pathways. In the 
absence of microtubules, Golgi membrane proteins would accumulate at peripheral ER exit sites 
due to failure of these structures to cluster into the centrosomal region. Regeneration of Golgi 
stacks over time in these peripheral sites would re-establish secretory flow from the ER to the 
Golgi and underlie the Golgi fragmentation process. 






Z01-HD-01 609-04 

Publications: 

Cole N and Lippincott-Schwartz J. Organization of organelles by microtubules. Current 
Opinion in Cell Biology 1995;7:55-64. 

Lippincott-Schwartz J. The Endoplasmic Reticulum-Golgi Membrane System. In: The 
Liver: Biology and Pathobiology, 3rd ed. New York: Raven Press, 1994;215-228. 

Lippincott-Schwartz J. Membrane traffic and compartmentalization within the secretory 
pathway. In: Schultz et al, eds. Molecular Biology of Membrane Transport Disorder. 
Plenum Press, 1995 (In press). 

Lippincott-Schwartz J, Cole N, Marotta A, Conrad PA and Bloom GS. Kinesin is the 
Motor for Microtuble-mediated Golgi-to-ER Membrane Traffic. J Cell Biol 1995; 128:293- 
306. 

Lippincott-Schwartz J and Cole N. Roles for microtubules and kinesin in membrane 
traffic between the ER and the Golgi complex. Biochemical Society Transactions 
1995;23:542-546. 

Lujan H, Marotta A, Mowatt MR, Sciaky N, Lippincott-Schwartz J and Nash TE. 
Developmental Induction of Golgi structure and function in Giardia lamblia. J Biol Chem 
1995;270:4612-4618. 

Sciaky N and Lippincott-Schwartz J. Cell Biological applications of confocal microscopy. 
Trends in Cell Biol 1994;4:227-228. 



tM] 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl-HD-01610-03 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Intracellular Metal Metabolism 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: Richard D. Klausner, M.D. 

Others: A. Dancis, Expert, CBMB 
R. Binder, NRC, CBMB 
A. Finegold, NRC, CBMB 
Y. Iwai, Visiting Fellow, 



Chief, CBMB 



CBMB 



R. Stearman, Guest Researcher, CBMB 
D. Yuan, Medical Staff Fellow, CBMB 



C. Philpott, Medical Staff Fellow, CBMB 



COOPERATING UNITS (if any) 

Anthony Segal, Dept.of Medicine, University College, London, UK; Teresa Dunn, 
of Biochemistry, USUHS, Bethesda, MD. 



Dept . 



LAB/BRANCH 

Cell Biology and Metabolism Branch 



SECTION 

Section on Organelle Receptor Structure and Function 



INSTITUTE AND LOCATION 

NICHD, Bethesda, MD 



TOTAL STAFF YEARS: 
5.25 



PROFESSIONAL: 
5.25 



OTHER: 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 

n (a1) Minors 
n (aP) Intfii-viRWR 



n (b) Human tissues H (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

In order to be incorporated into essential cellular enzymes, iron must first traverse various membrane barriers, and yet the 
molecular mediators of these transport processes have not been identified. We have utilized the genetically tractable eukaryote, 
Saccharomyces cerevisiae, as a model to identify genes required for the high-affinity transport of iron across the plasma 
membrane. These genes include CTR1 , CCC2, FET3 and FTS3. The FET3 gene encodes an oxidase activity required for 
high-affinity iron uptake, and the amino acid sequence exhibits similarity to the multi-copper oxidases laccase, ascorbate oxidase 
and, to some extent, ceruloplasmin. IVIutations in CTR1 or CCC2 result in the production of an inactive FET3 apoprotein due to 
failure to incorporate copper, and the defect in iron uptake in these mutants is a consequence of deficiency of FET3 oxidase 
activity. Genetic evidence of interaction of FET3 and FTS3 has been obtained: 1) overexpression of both genes is required to 
augment iron uptake in a wild-type strain, 2) deletion of FTS3 results in loss of iron uptake activity and FET3 oxidase function, 
and 3) specific mutant alleles of FTS3 abrogate iron uptake without effect on oxidase function. Thus, FET3 may act together 
with FTS3, which encodes a polytopic membrane protein, to form an iron transporting complex. A genetic approach has also 
enabled us to identify the iron regulator, AFT1 , which mediates the homeostatic control of the iron uptake system. This is 
accomplished through interaction of the AFT1 protein with cis-acting elements in promoter DNA under conditions of iron 
deprivation, inducing transcription of the target genes in an iron dependent fashion. 



84 



PHS 6040 (Rev. 5/92) 



ZOl-HD-01610-03 

INTRACELLULAR METAL METABOLISM 

Objectives 

(1) To use the methodology of yeast genetics to identify genes 
involved in the regulated uptake and utilization of iron, (2) to 
elucidate the mechanism of the transmembrane transport of iron, 
and (3) to determine how cellular iron status is "sensed" and how 
iron-dependent gene regulation ensues from this. 

Major Findings 

FET3 - Explaining the Copper Dependence of Iron Uptake 

Cells that are severely deprived of copper exhibit deficiencies 
of copper enzymes, for example, copper-zinc superoxide dismutase. 
Such copper starved cells also exhibited deficient high affinity 
iron uptake. The physiologic basis for this deficiency was 
suggested by the observation that FET3 , a gene required for high 
affinity iron uptake, contains sequence motifs that resemble the 
copper-coordinating amino acids of a multi-copper oxidase. A 
biochemical assay for the FET3 oxidase was developed in the lab, 
and using this assay, it was possible to directly demonstrate a 
copper requirement for the oxidase activity. Thus, the copper 
requirement for iron uptake stemmed from the need to provide 
copper to FET3 protein, a copper-containing oxidase required for 
iron uptake . 

The FET3 oxidase assay also provided a means to identify FET3 
apoprotein. In cells deprived of copper, FET3 protein is 
synthesized and is stable, but it has no activity. Activity could 
be restored to the apoprotein by addition of copper to the cell 
lysate. As described in the previous Annual Report, we 
identifed two other genes required for high affinity iron 
uptake. CTRl was identified by means of the FRE1-HIS3 selection 
scheme and encodes a copper transport protein localized to the 
plasma membrane and functioning in copper acquisition from the 
environment. CCC2 was identifed by utilizing degenerate 
oligonucleotides derived from conserved domains of P-type ATPases 
to amplify related gene sequences from S. cerevisiae genomic DNA. 
The sequence of CCC2 exhibits strong and global similarity to the 
family of copper-transporting P-type ATPases, including the human 
genes implicated in Wilson disease and Menkes disease. Mutation 
or deletion of CTRl or CCC2 resulted in failure of adequate 
copper to reach newly synthesized FET3 protein. Only inactive 
apoprotein was produced, and consequently, iron uptake was 
deficient in these mutants. 

Additional indirect evidence suggested to us a model in which 
CTRl mediates copper transport into the cell, and CCC2 mediates a 
second copper transport step across an internal cellular 



.S5 



ZOl-HD-01610-03 

membrane. Both of these transporters must function in series in 
order for copper loading of FET3 protein to occur. The evidence 
for this model derives from data indicating that CTRl mutants 
were deficient in cellular copper uptake whereas CCC2 mutants 
were not. Thus, the existence of apo-Fet3p in CCC2 mutants 
derived from a copper handling defect hoccurring after uptake of 
copper into the cell. In addition, we noted that the FET3 
protein contained N-linked sugars as evidenced by EndoH 
sensitivity. Analysis of the sequence of the predicted FET3 
protein revealed a single transmembrane domain and predicted 
N-glycosylation sites on the same side of the membrane as the 
copper coordination sites. Therefore, copper delivery to the 
FET3 protein must require a membrane crossing step to gain 
entrance into the secretory pathway, where both sugar addition 
and copper insertion are likely to occur. Mediation of this step 
in copper delivery is a likely site of action for the CCC2 
P-type ATPase. This function is analogous to the proposed 
function of the Menkes and Wilson disease gene products. In the 
latter case, the copper- transporting P-type ATPase functions to 
deliver copper into the secretory pathway, where it is inserted 
into ceruloplasmin, a multi-copper oxidase that is secreted into 
the blood. Interestingly, mutations in the ceruloplasmin gene 
in humans have recently been linked to disorders of iron 
homeostasis . 

Identification of FTS3 . a Candidate Gene for the Iron Transporter 

A biological problem that has preoccupied us for some time is the 
question of how iron is transported across membranes. Although we 
were able to identify various mutants with deficiency of high 
affinity iron uptake, the corresponding genes did not include 
the iron transporter. CTRl and CCC2 encode copper transporters, 
and FET3 encodes a membrane-associated oxidase with no 
resemblance to the family of permeases. A likely candidate for 
the iron transporter was discovered in the lab by investigating 
the properties of E3 0, an unusual mutant identified by means of 
the FRE1-HIS3 selection. This mutant exhibited recessive 
phenotypes that included high levels of ferric reductase 
activity, high ferrous uptake activity (5-10 times normal) and 
slow growth which could not be rescued by iron addition to the 
medium. The slow growth of the mutant resulted in genetic 
instability and the generation of visible papillae on all types 
of growth media. We isolated clones from these papillae and 
found that in some cases they exhibited more rapid growth and 
severely depressed iron uptake. The basis for the depressed iron 
uptake was analyzed and found to be the result of second site 
mutations. When these second site mutations were analyzed 
genetically, we found that several loci were represented: CTRl, 
FET3 , and an unidentifed locus. The wild-type copy of the unknown 
mutated gene was then cloned by complementation. In order to 
achieve this, we used the chelator, ferrozine, to limit iron in 



.•^G 



ZOl-HD-01610-03 

the growth medium, thereby allowing the wild- type or 
complemented mutants to grow and inhibiting growth of the strains 
with defective iron uptake activity. The complementing gene was 
called FTS3 , for Ferrous Transport Suppressor 3, and a marked 
allele of this gene was reintegrated into the genome. The 
absence of meiotic recombination between the marked allele and 
the original mutation demonstrated that we had cloned the correct 
gene. The complementing activity was contained within an open 
reading frame with 6 predicted hydrophobic regions consistent 
with transmembrane domains. A domain was also noted with sequence 
similarity to a Bacillus Subtilis permease of unknown function. 
However, the 5' region of the homologous bacterial gene contained 
an an excellent pallindromic recognition site for the iron 
regulator Fur. Finally, we observed that the FTS3 transcript was 
iron regulated in a manner consistent with its presumed role in 
uptake i.e. the transcript was induced by iron deprivation and 
repressed by iron replete growth conditions. FTS3 , then, appeared 
to be an excellent candidate gene for the cellular iron 
transporter . 

Genetic Evidence for FTS3/FET3 Association 

The phenotypes of FTS3 and FET3 mutants were noted to be similar: 
each was incapable of growth on f errozine-containing medium and 
lacked high affinity iron uptake under all growth conditions. 
This observation raised the possibility that the FET3 and FTS3 
proteins might interact, either directly or indirectly. Moreover, 
we noted that expresssion of FET3 from a multi-copy plasmid, 
which conferred increased oxidase expression, did not result in 
increased iron uptake activity. Only when FTS3 and FET3 were 
overexpressed together was iron uptake increased above the 
wild-type level, perhaps due to a requirement for both genes in 
mediating iron uptake. 

Additional evidence for interaction of FTS3 with FET3 was 
obtained by examining the effect of mutation of FTS3 on the FET3 
oxidase. Complete deletion of the FTS3 gene abrogated oxidase 
activity, although the amount of FET3 protein synthesized was 
unaffected. The defect appeared to be due to the failure of 
copper loading of the oxidase protein, since some degree of 
restoration of oxidase activity could be achieved in vitro by 
adding copper to the lysate. Three possible explanations for 
this finding were considered: 1) FTS3 functions in the 
delivery of copper to FET3 (like CCC2 ) ; 2) FTS3 transiently 
associates with FET3 in order to deliver it to the correct 
cellular compartment, where it then acquires copper (chaperone 
function); or 3) FTS3 forms a stable association with FET3 which 
is required for the correct localization of both gene products. 
This association is required for copper loading of the oxidase 
and for iron transport . 



87 



ZOl-HD-01610-03 

Evidence of a Direct Role of FTS3 in Iron Uptake 

We have been able to eliminate the first two possible 
explanations mentioned above by identifying mutant alleles of 
FTS3 that interfere with iron uptake, while permitting normal 
expression, copper loading and full activation of the FET3 
oxidase. Truncation of the 3' end of the FTS3 open reading frame 
by insertion of a stop codon at a unique BstXI site (removing 70 
amino acids from the carboxy terminus ) blocks iron uptake 
without affecting oxidase activity. Removal of an additional 54 
amino acids by truncating at a unique EcoRV site abolishes 
oxidase activity and iron uptake. In addition, several 
site-directed mutants were constructed in glutamic acid 
residues, singled out because of their occurrence in a conserved 
motif: REGLE . This motif occurs in slightly altered form in the 
ferritin light chain sequence: REGAE. Structural data suggests 
that the E residues in this motif are involved in interaction 
with iron at the nucleation site in the interior of the ferritin 
shell. We constructed corresponding mutations in the FTS3 
protein and found that they inhibit iron uptake without affecting 
oxidase function. Thus these mutations provide additional 
evidence that FTS3 protein interacts directly with iron. In a 
sense, the FTS3 protein can be considered to be bifunctional, 
since a portion of the protein is required for copper 
acquisition (and probably correct targeting) of FET3 , while 
another portion of the protein is required for iron uptake. 

To serve as the cellular iron uptake transporter, the FTS3 
protein must reside on the cell surface. We sought to 
demonstrate this directly by placing a myc epitope in frame at 
the 3' end of the FTS3 open reading frame. The tag insertion did 
not interfere with the ability of the clone to complement an FTS3 
deletion strain, and the protein could be visualized as a bright 
rim of immunof luoresence at the periphery of the cell, consistent 
with a plasma membrane localization. We are now actively 
striving to demonstrate physical association between the FTS3 and 
FET3 proteins in order to complete the picture of an FTS3/FET3 
iron transporting complex. 

The mechanism of iron transport suggested by these emerging 
molecular details involves what at first appears to be a paradox. 
The FREl reductase reduces iron chelates outside the cell, and 
the FET3 oxidase reoxidizes the iron. However, the many caveats 
to this statement make it seem less paradoxical. The reduction of 
iron is not directly coupled to transport; iron reduction at the 
cell surface, which may be necessary to mobilize ferric iron 
chelates, occurs in great quantitative excess over the iron 
transport activity. For example, nanomole quantities of iron are 
reduced per million cells versus only picomole quantities that 
are taken up. Subsequently, the reduced iron likely interacts 
with the FET3/FTS3 complex. The oxidase activity of this complex 



.•^8 



ZOl-HD-01610-03 

appears to be coupled to the movement of iron across the 
membrane. Although the substrate for the oxidase has not been 
definitely ascertained, it is possible that the oxidase acts on 
residues in the transporter protein that then interact with the 
transported iron. Alternatively, the FET3 oxidase might act 
directly on the ferrous iron, generating a species of iron that 
is transport competent. Work on ferritin has shown that it 
possesses a ferroxidase activity which generates ferric monomers 
that can then be transported from one ferritin polymer to 
another. The transported iron species, ferric monomer, is only 
transiently formed upon reoxidation of ferrous iron in proximity 
to a protein surface. Analogously, the presence of the transport 
protein surface (FTS3 protein) in proximity with the oxidase 
(FET3 protein) could be necessary to directly interact with the 
ferric iron species that is formed, perhaps through critical 
carboxlate residues. This process must compete with the process 
of ferric iron hydration and precipitation that occurs in 
acqueous solutions . 

Cellular Iron Homeostasis - Role and Mechanism of Action of AFTl 

Iron is required for incorporation into essential metalloproteins 
utilized for electron transfer, redox chemistry and other 
cellular functions, but excess iron is toxic. Homeostatic 
regulation of the cellular iron status, therefore, is important 
to cell survival. In yeast, this objective is achieved through 
the transcriptional regulation of essential components of the 
uptake system. We have learned that this regulation affects not 
just one rate limiting component but all the components of the 
iron uptake system thus far identified. In previous Annual 
Reports, we noted iron regulation of FREl and FRE2 , the genes 
encoding the surface reductase. Also, FET3 , the copper dependent 
oxidase was found to be iron regulated. This year, we have 
observed similar coordinate, iron dependent regulation of CCC2 , 
the copper transporting P-type ATPase and FTS3 , the putative iron 
transporter. Two additional iron regulated genes were identified 
by means of a genetic screen originally devised by M. Snyder. In 
this scheme, yeast genomic DNA was marked with random lacZ 
insertions by means of Tn3 "hops" and then reintegrated into 
the genome of a diploid yeast strain. These lacZ insertion 
mutants were screened for regulated lacZ activity on iron rich 
versus iron poor plates. The two genes identified by this 
approach include one with strong homology to the MDR family of 
ATP-dependent pumps (distinct from the P-type ATPase family) . 
Determination of the localization and function of the gene 
product has not yet been achieved, but conceivably it could serve 
an iron pumping function across an internal cellular membrane. 

For each of the iron regulated genes identified, the regulation 
occurs at the level of transcription and is mediated by AFTl, the 
iron regulator gene which we identifed and cloned last year (see 



.SJ] 



ZOl-HD-01610-03 

previous Annual Report) . The identification of the dominant 
AFTl-lup allele, in which the expression of iron regulated genes 
is constitutive, and the construction of an interrupted 
(loss-of-function) allele, in which expression of these genes is 
severely impaired, led us to believe that AFTl must be an 
activator of transcription. This led to the prediction that, 
under under starvation conditions, AFTl should interact either 
directly or indirectly with the promoter regions of iron 
regulated genes. By analysis of gene fusions, the cis-acting 
element mediating AFTl regulation has been defined, and direct 
interaction of the AFTl protein with this element has been 
demonstrated utilizing a gel mobility shift assay. In addition, 
by means of an in vivo footprinting assay, the regulated 
interaction of AFTl with its target element on the yeast genome 
has been demonstrated. In view of this data, it is likely that 
AFTl mediates iron regulation of transcription through direct 
interaction with the promoter DNA of its target genes. 

The sensing of cellular iron levels may also be mediated through 
AFTl. The AFTl-lup allele contains a mutation that alters the 
cysteine to a phenylalanine at position 291 of the protein. 
Perhaps this critical cysteine residue together with other 
cysteines in the AFTl protein coordinates iron when the cell is 
iron replete, stabilizing an AFTl protein conformation which is 
incapable of interacting with DNA. In a situation in which iron 
is limited, perhaps the AFTl protein switches conformation and 
binds to DNA, thereby initiating transcription. Defining the 
mechanism of such a transcriptional regulatory switch will 
require further genetic and biochemical work. 

The toxicity of iron is thought to be mediated by the 
participation of iron in free radical generation in the presence 
of oxygen. Free radicals are thought to damage vital cellular 
macromolecules . However, other mechanisms of toxicity, such as 
misincorporation of iron in zinc proteins, could also be 
important. Moreover, the specific cellular targets of iron 
overload toxicity are not known in any detail, nor are the 
cellular defense mechanisms well characterized. To address some 
of these questions genetically we have initiated a screen for 
mutants which are synthetically lethal with the AFTl-lup allele, 
expressed under the control of the GallO promoter. Mutants have 
been identiied and are being characterized. In addition, 
candidate genes such as the RAD genes that might protect against 
iron toxicity are being evaluated for the synthetic lethal 
effects when combined with the AFTl-lup allele. 

Relevance to Human Physiology and Disease 

The genetic analysis of the iron uptake system in yeast has 
continued to yield insights that may be relevant to human 
physiology and disease. This relevance can be viewed on two 



40 



ZOl-HD-01610-03 

levels. On one level, the yeast genes are homologous to human 
genes to varying degrees. FREl , the structural gene for the yeast 
iron reductase is homologous to gp91-phox, the structural gene 
for the human granulocyte respiratory burst oxidase. The visible 
spectra of these two membrane cytochromes are virtually 
super imposable. As part of a collaboration with Anthony Segal, 
we have successfully overexpressed the yeast protein under the 
ADHl promoter and placed an epitope tag that allows us to analyze 
protein expression. He will determine the midpoint potential of 
the yeast protein by titrating the FREl heme spectrum. This 
may shed light on the mechanism of action and substrate 
specif icty of the reductase. Domain swapping with the human 
protein may yield structure-function information regarding such 
issues as heme coordination and subunit interaction, information 
which has been difficult to ascertain for the human multi-subunit 
complex. Other areas of relevance to human disease stem from the 
CCC2 homology to the human Menkes and Wilson disease genes. 
Areas that could be purusued relate to the analysis of human 
disease mutations in the yeast system (corresponding mutations 
could be made in the yeast genes) . Localization of the yeast 
gene product might have implications for the mechanism and the 
cellular site of action of the Menkes and Wilson disease gene 
products. 

On a more general level, the linkage of cellular copper and iron 
homeostasis observed in yeast is apparently conserved in humans. 
Copper deficiency, mutation in the Wilson disease gene or 
mutation in the ceruloplasmin gene can lead to severe 
perturbations in iron physiology. The description of kindreds 
with ceruloplasmin mutations and abnormal iron handling has only 
recently been reported. Finally, if FTS3 is the iron transporter 
of yeast, this raises the question of whether an FTS3 homologue 
exists in humans. If ceruloplasmin is the human FET3 homologue 
and the mechanism of iron transport requires interaction with a 
multicopper oxidase, then how might this ceruloplasmin interact 
with a human iron transporter? Identification of the human iron 
transporter would be of great interest because of the relevance 
to diseases of iron metabolism, such as hemochromatosis. This is 
a common genetic disease resulting from the failure of the 
homeostatic regulation of iron uptake from the gut. The genetic 
locus has been known to reside in the HLA region of Chromosome 6 
but the gene has still not been cloned, due to the lack of 
recombinants in this region. More generally, the human iron 
transporter gene would be of importance because of its effects 
on general processes that require iron i.e. cell proliferation, 
respiration, metabolism and defense. 



41 



ZOl-HD-01610-03 



Publications 



Anderson GJ, Dancis A, Roman DG, and Klausner RD. Ferric iron 
reduction and iron uptake in eucaryotes : studies with the yeasts 
Saccharomyces cerevisiae and Schizosaccharomyces pombe . Adv Exp 
Med Biol 1994;356:81-89. 



Dancis A, Haile D, Yuan DS, and Klausner RD. The Saccharomyces 
cerevisiae copper transport protein (Ctrlp) : Biochemical 
characterization, regulation by copper and physiologic role in 
copper uptake. J Biol Chem 1994;41:25660-25667. 



Klausner R and Dancis A. A genetic approach to elucidating 
eukaryotic iron metabolism. FEES Letters 1994;355:109-113. 



Yamaguchi-Iwai Y, Dancis A, and Klausner RD. AFTl : A mediator of 
iron regulated transcriptional control in Saccharomyces 
cerevisiae. EMBO J 1995;14:1231-1239. 



Yuan DS, Stearman R, Dancis A, Dunn T, Beeler T, and Klausner RD. 
The Menkes/Wilson disease gene homologue in yeast provides copper 
to a ceruloplasmin-like oxidase required for iron uptake. Proc 
Natl Acad Sci USA 1995; 92:2632-2636. 



42 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl-HD-01611-01 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

The VHL Tumor Suppressor Gene 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
PI: Richard D. Klausner, M.D., Chief, CBMB, NICHD 

D.Chen, HHMI, CBMB, NICHD 

S. Lee, Visiting Fellow, CBMB, NICHD 

A. Pause, Visiting Fellow, CBMB, NICHD 



COOPERATING UNITS (if any) 

NCI, Surgery Branch (Roxanne Duan, James Gnarra, W. Marston Linehan) and the 
Okalahoma Medical Research Foundation (Drs. Ronald and Joan Conaway) 



LAB/BRANCH 

Cell Biology and Metabolism Branch 



SECTION 

Section on Organelle Receptor Structure and Function 



INSTITUTE AND LOCATION 

NICHD, Bethesda, MD 



TOTAL STAFF YEARS; 

3 .25 



PROFESSIONAL: 
2.25 



OTHER: 
1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 

n (a1) Minors 
n (aP) Intprvifiw/R 



n (b) Human tissues Kl (c) Neitlner 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The VHL gene was first identified in 1993 as the gene responsible for a rare inherited 
cancer syndrome called Von Hippel-Lindau disease. Inheritance of a single defective 
VHL gene results in the predisposition to the development of a variety of cancers 
including clear cell renal carcinoma, pheochromocytoma, and angioblastomas of the 
central nervous system, among others. Subsequently, it was shown that the majoritii 
of tumors derived from patients with sporadic clear cell renal carcinoma alsc 
contained both loss of heterozygosity of the VHL locus and failure of expression 
and/or mutations in the remaining VHL allele. The goal of this project is tc 
understand the biochemistry and cell biology of the VHL gene product and thereby tc 
elucidate its role in cell transformation. Studies over the past year include the 
completion of the cloning and sequencing of the human and rat VHL gene, the 
biochemical characterization of the gene product and its intracellular localization. 
The identification and cloning of gene products that VHL interacts with has 
illuminated an unexpected new area of gene control--the regulation of transcriptional 
elongation. 



43 



PHS 6040 (Rev, 5/92) 



ZOl-HD-01611-01 
THE VHL TUMOR SUPPRESSOR GENE 

Findings 

Cloning and Comparison of the Rat and Human VHL cDNAs 

Six independent rat cDNA clones were obtained by hybridization with human VHL cDNA using 
sequences derived from EXONs 1 and 2. The longest rat cDNA of approximately 2.81 Kb 
hybridized to a single mRNA species from rat tissues and cell lines of 2.91 Kb in length. In contrast 
to the human protein, the presence of upstream in-frame termination codons allowed us to uniquely 
assign a start codon for the rat gene. The fact that amino acid nucleotide homology between the rat 
and human gene begins with the rat start codon allowed us to finally predict the assignment of the 
human start codon. The rat VHL cDNA predicts a protein of 185 amino acids compared to the 
human protein of 213 amino acids. The size difference between the two proteins results largely from 
a single region of disparity near the amino terminus. In the human protein a unique gly-x-glu-glu-x 
acidic motif is repeated eight times, while this sequence occurs only once in the rat protein. Aside 
from the acidic repeats, the predicted rat VHL protein shares 88% sequence identity with the 
corresponding sequence in the predicted human VHL protein. 

Subcellular Localization of VHL 

Epitope tagged human and rat proteins were introduced into several cell lines and examined by 
immunofluorescence microscopy. The surprise finding was that in any population of transiently 
transfected cells, a variety of patterns were observed. Many cells that are predominantly or uniquely 
nuclear localization while other cells had a cytosolic localization with sparing of the nucleus. Finally, 
a population of cells appear to have localization in both nucleus and cytosol. This varied distribution 
of the protein was confirmed by subcellular fractionation. 

Formation of Protein Complexes 

When VHL was either transiently or stably expressed in a variety of recipient cell types it was 
observed to associate with numerous endogenous proteins. This was assessed by steady state 
metabolic labeling followed by immunoprecipitation using antibodies against the epitope tag 
introduced into either the amino or carboxy termini of either the rat or human VHL cDNAs. 
Numerous proteins were thus observed specifically assembling with VHL. These included a 
pentamer of proteins between 55 and 60 kD and two proteins of approximately 9 and 16 kD. 
Sucrose gradient centrifiigations separated these into several complexes including one that appeared 
to contain stoichiometric amounts of VHL and the 9 and 16 kD proteins. When a variety of naturally 
occurring point mutations, observed either in tumors from sporadic kidney cancer or from the germ 
line of VHL patients were examined, they demonstrated a complete or partial loss of the ability of 
VHL protein to specifically assemble with the 9 and 16 kD partners. 



44 



ZOl-HD-01611-01 

Purification and Identification of VHL Associated Proteins 

The specific loss of the 9 and 16 kD proteins with presumably inactivating VHL mutations led us to 
purify these two proteins. This was accomplished by the creation of stable transfectants of epitope 
tagged VHL followed by immunoprecipitation after large scale cell culturing. Immunoaffinity 
purification followed by SDS page electrophoresis resulted in the purification of sufficient amounts 
of the 9 and 16 kD proteins to allow peptide sequencing. Each of these proteins were digested with 
a protease (lysC) and the peptides eluted and separated by HPLC for sequential Edman degradation. 
The protein sequences were thus determined. The 16 kD protein proved to be identical to a protein 
in the database which is a subunit of an RNA polymerase (poll!) elongation factor referred to as SIII 
or elongin. Elongin is a heterotrimer consisting of three proteins: elongin A (1 10 kD), elongin B 
(18 kD), and elongin C (14 kD). The 16 kD VHL associated protein is identical to elongin B. 
Sequencing of the 9 kD protein demonstrated that it was identical to the unpublished sequence 
(received fi-om Ron and Joan Conaway) of elongin C. We used antibodies specific for elongin B and 
C to demonstrate that indeed the VHL associated proteins were these two subunits of the elongation 
complex. 

Assembly of VHL with Elongin Subunits 

We used a variety of techniques to establish the ability of VHL protein to assemble in vitro with 
elongin subunits. These included the use of purified bacterially produced recombinant proteins or 
the use of in vitro transcription/translation coupled reactions. For these studies, we utilized and 
examined all possible interactions between VHL and elongins A, B, and C. These studies 
demonstrated that VHL was not capable of assembling with elongin A, regardless of the presence of 
other elongin subunits. There is some binding of VHL to elongin C in the absence of any other 
subunit, while there was no detectable interaction of elongin B with VHL. When elongin B and C 
were both present, a large amount of assembly with VHL was seen with the apparent production of 
a trimer of stoichiometry 1:1:1. This was the same pattern of assembly that was seen between 
elongin A and the B and C subunits. Elongin B and C share no sequence similarities with VHL, but 
VHL does contain a 13 amino acid stretch that is very similar (10 out of 13 identicals) to a sequence 
found in elongin A. A peptide containing this sequence derived from VHL is capable of competing 
for the assembly of either elongin A or VHL with elongin B and C. 

Effect of VHL on Elongin-Stimulated Transcriptional Elongation 

To investigate whether VHL affects elongin activity, we included VHL in two different assays of 
transcriptional elongation: (1) the adenovirus 2 major late promotor runoff transcription assay and 
(2) the oligo(dC)-tailed template assay which permits direct measurement of elongin activity in the 
absence of general initiation factors. When VHL was titrated into such reactions in the presence of 
the three elongin subunits there was a dose-dependent complete inhibition of poll! transcription 
elongation activity. This inhibition could be quenched by the titration of elongin B and C subunits. 
All of these data demonstrate that VHL can compete with elongin A for assembly with elongin BC 
in such a way VHL appears to be capable of regulating transcription at the level of polll elongation. 



4 



r: 



ZOl-HD-01611-01 

Publications 

Duan DR, Humphrey JS, Chen DYT, Weng Y, Sukegawa J, Lee S, Gnarra J, Linehan, 
WM, and Klausner RD. Characterization of the VHL tumor suppressor gene 
product: Localization, complex, formation, and the effect of natural 
inactivating mutations. PNAS 1995;92:6459-6463. 

Duan DR, Pause A, Burgess WA, Aso T, Chen DYT, Garrett KP, Conaway RC, Conaway 
JW, Linehan WM, and Klausner RD. Inhibition of transcription elongation by the 
VHL tumor suppressor protein. Science 1995;269:1402-1406. 



46 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00610-15 DEB 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 



Growth, Puberty, Their Disorders: Physiology, Pathophysiology, and Molecular Biology 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

P.I.: G.B. Cutler Head SDE, DEB, NICHD 

Others: (see attached) 



COOPERATING UNITS (if any) 

(see attached) 



LAB/BRANCH 

Developmental Endocrinology Branch 



SECTION 

Section on Developmental Endocrinology 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
7.4 



PROFESSIONAL: 

6.1 



OTHER: 



1.3 



CHECK APPROPRIATE BOXIESI 

E (a) Human subjects 
E (a1) Minors 
D (a2) Interviews 



E (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The objective of this project is to advance understanding of the mechanisms that underlie normal and abnormal 
puberty, and to apply this knowledge to improve existing therapy for disorders of puberty. Since somatic growth is a 
major determinant of the timing of pubertal onset, a further objective is to clarify the mechanisms of normal growth 
and of growth failure. Principle areas of laboratory investigation include the mechanisms of normal growth and 
puberty. Principle areas of clinical investigation include the mechanism of premature thelarche and of the 
gonadotropin-independent forms of precocious puberty, the developmental changes in hypothalamic regulation of 
gonadotropin secretion, the behavioral changes associated with normal and abnormal pubertal development, the 
mechanisms of prepubertal and pubertal growth, the role of pubertal sex steroids in the acquisition of normal adult 
bone density, the treatment of central precocious puberty with an analog of luteinizing hormone-releasing hormone 
(LHRH), the treatment of familial male isosexual precocious puberty with combined antiandrogen and aromatase 
inhibitor, the evaluation of new approaches to the diagnosis of growth hormone deficiency and to the differential 
diagnosis of delayed puberty, the treatment with synthetic parathyroid hormone of children with hypoparathyroidism, 
the treatment with growth hormone of children with Turner syndrome and with non-growth hormone-deficient short 
stature, and the treatment with insulin-like growth factor- 1 of children with growth hormone insensitivitv and of 
children with non-growth hormone-deficient short stature. 

The principal areas of laboratory investigation include the structure and function of the regulatory sequences of the 
human gonadotropin-releasing hormone (GnRH) gene, molecular analysis of the gene for the luteinizing hormone 
receptor in familial male precocious puberty and in Leydig cell hypoplasia, and of the gene for the calcium-sensing 
receptor in sporadic and autosomal dominant hypoparathyroidism, and hormonal regulation of epiphyseal 
transforming growth factor-B. fibroblast growth factor, platelet-derived growth factor, and the growth hormone 
receptor. To examine the effects of these growth factors in vivo we are studying rabbits bearing small needles 
implanted into the proximal tibial epiphyses and connected to osmotic minipumps. 



PHS 6040 (Rev. 5/92) 



00 



ZOl HD 00610-15 DEB 



Others: 



K.M. Barnes 


Chemist 


SDE, 


DEB, 


NICHD 


J. Baron 


Senior Clinical Associate 


SDH, 


DEB, 


NICHD 


D. Counts 


Special Volunteer 


SDE, 


DEB, 


NICHD 


F. DeLuca 


IRTA Fellow 


SDE, 


DEB, 


NICHD 


A. Cunningham 


Pre-IRTA Fellow 


SDE, 


DEB, 


NICHD 


F. Czerwiec 


Clinical Associate 


SDE, 


DEB, 


NICHD 


P. Feuillan 


Special Volunteer 


SDE, 


DEB, 


NICHD 


N. Gerstle 


Special Volunteer 


SDE, 


DEB, 


NICHD 


P. Gore 


Summer IRTA 


SDE, 


DEB, 


NICHD 


B. Hossein-Zadeh 


Summer IRTA 


SDE, 


DEB, 


NICHD 


C. Heinrichs 


Special Volunteer 


SDE, 


DEB, 


NICHD 


L. Laue 


Adjunct Scientist(IPA-G'town) 


SDE, 


DEB, 


NICHD 


E. Leschek 


Clinical Associate 


SDE, 


DEB, 


NICHD 


K. Oerter-Klein 


Special Volunteer 


SDE, 


DEB, 


NICHD 


S. Rose 


Special Volunteer 


SDE, 


DEB, 


NICHD 


J. Levine Ross 


Special Volunteer 


SDE, 


DEB, 


NICHD 


K. Winer 


IRTA Fellow 


SDE, 


DEB, 


NICHD 


K. Yano 


Visiting Associate 


SDE, 


DEB, 


NICHD 


J. Yanovski 


Clinical Associate 


SDE, 


DEB, 


NICHD 



Cooperating Units 

J. Jones, L. Long, D. Blum, S. Hill, A. Dwyer, T. Shawker, Clinical Center, NIH; M. Bolander, Mayo Clinic; A. 
Hidaki, M. Saji, L.D. Kohn, Laboratory of Biochemistry and Metabolism, NIDDK; W.-Y. Chan, S.-M. Wu, L. 
Blomberg, Department of Pediatrics, Georgetown University; A.J.W. Hsueh, M. Kudo, S.Y. Hsu, Department of 
Obstetrics and Gynecology, Stanford University Medical Center. 



Ojl 



ZOl HD 00610-15 DEB 

Project Description 

Objectives: 

We seek to increase understanding of normal and abnormal growth and pubertal development, and to apply this 
knowledge to improve treatment for disorders of growth and puberty. The specific objectives are: 

1) to determine gonadotropin pulse frequency and amplitude during normal puberty, and to compare the normal 
pattern with the pattern in children with central precocious puberty, particularly children with a hypothalamic 
hamartoma; 

2) to identify the molecular mechanism of testicular autonomy in boys with familial male precocious precocity 
(FMPP), and of Leydig cell failure in Leydig cell hypoplasia, through analysis of the gene for the luteinizing 
hormone (LH) receptor; 

3) to explore the mechanism of premature thelarche; 

4) to develop improved methods for the diagnosis of growth hormone deficiency and for the differential diagnosis 
of delayed puberty; 

5) to determine the efficacy and safety of treating the gonadotropin-independent ovarian function of McCune- 
Albright syndrome with aromatase inhibitors; 

6) to determine the efficacy and safety of treating familial male precocious puberty with combined antiandrogen 
and aromatase inhibitor; 

7) to determine the efficacy and safety of LHRH analog therapy of central precocious puberty through long-term 
follow-up of children under treatment; 

8) to understand the mechanism of the pubertal growth spurt and its relationship to prepubertal growth, to 
optimize medically-induced prepubertal and pubertal growth in Turner syndrome through clinical trials of growth 
hormone (with and without low-dose estrogen treatment), to understand the mechanisms of non-growth hormone 
(GH)- deficient short stature and to improve the long-term outcome, and to assess the efficacy and safety of 
LHRH agonist-induced pubertal delay, of growth hormone treatment, and of treatment with insulin-like growth 
factor- 1 on adult height in children with non-GH-deficient short stature; 

9) to improve understanding of the role of estrogen in growth and development during childhood, early puberty, 
and the pubertal growth spurt by applying a newly developed ultrasensitive recombinant cell bioassay for 
estradiol; 

10) to examine the hormonal regulation in epiphyseal cartilage of transforming growth factor-P, fibroblast growth 
factor, platelet-derived growth factor, epidermal growth factor, and GH receptor; 

11) to understand the regulation of epiphyseal growth in vivo through the response to added growth factors and 
to blockade of growth factor action, using both classical physiologic approaches and the creation of transgenic 
animals; 

12) to explore the role of pubertal sex steroid secretion in the acquisition of normal adult bone density; 

13) to study the structure and regulation of the normal human LHRH gene and to compare the observations with 
the results in genetic disorders causing early or late puberty; to study the regulation of the LHRH gene and of 
LHRH secretion in a cultured LHRH neuronal cell line; 



0^ 



ZOl HD 00610-15 DEB 

14) to correct the absence of parathyroid hormone action on bone and kidney in patients with hypoparathyroidism 
by administration of synthetic parathyroid hormone; 

15) to identify the molecular mechanism of autosomal dominant and sporadic hypoparathyroidism through analysis 
of the gene for the calcium-sensing receptor. 

Methods Employed: 

l)Hypothalamic-pituitary maturation during puberty-steroid and peptide hormone radioimmunoassay andbioassay. 

2) Mechanism of familial male precocious puberty and of Leydig cell hypoplasia - molecular studies of the LH 
receptor gene by PCR, sequencing, site-directed mutagenesis, and expression in cultured cells. 

3) Treatment of precocious puberty - clinical, radiographic, behavioral, and hormonal evaluation. 

4) Mechanism of normal prepubertal and pubertal growth, and of idiopathic growth failure - bioassay and 
radioimmunoassay of growth factors; immunostaining of peptide growth factors in epiphyseal cartilage; in situ 
hybridization, solution hybridization, and/or Northern analysis to quantitate growth factor mRNAs in epiphyseal 
cartilage; infusion of growth factors or growth factor antagonists into the rabbit proximal tibial epiphyseal growth 
plate; creation of transgenic animals from embryonic stem cells with heterozygous or homozygous cartilage- 
specific deletion of growth factors; molecular studies of the growth hormone (GH) receptor (GHR) gene. 

5) Treatment of growth failure - randomized, double-blind, placebo-controlled trials with aduh height as the 
principal growth endpoint; short-term studies to define the conditions that appear optimal for long-term evaluation. 

6) Mechanism of autosomal dominant or sporadic hypoparathyroidism - molecular studies of the calcium-sensing 
receptor gene by PCR, sequencing, site-directed mutagenesis, and expression in cultured cells. 

Progress: 

1. Mechanism and treatment of precocious puberty 

a. Familial male precocious puberty (FMPP) and Leydig cell hypoplasia - molecular mechanism 

To determine the structural requirements for activating mutations of the LH receptor (LHR), the mutations were 
identified from more than 40 families with familial male precocious puberty (FMPP) from widely separated 
geographic regions. A total of 8 different mutations were identified, which comprise 2/3 of the 12 known 
activating mutations of the LHR. Eleven of the 12 activating mutations are in a 40-amino acid region from 
position 542 to 581, which spans the fifth and sixth transmembrane domains and the intervening third intracellular 
loop. This hot spot for activating mutations identifies a region that plays a critical role in constraining the 
unliganded receptor in an inactive conformation. Thus, the studies of this rare form of precocious puberty have 
pinpointed a region of the LH receptor that is critical for signal transduction and hence for the hormonal 
regulation of human reproduction. 

Leydig cell hypoplasia is an autosomal recessive form of male pseudohermaphroditism. To test the hypothesis 
that this disorder results from inactivating mutations of the LH receptor, we performed studies similar to those 
described above for FMPP. To date several mutations have been identified that appear to explain this disorder: 
(1) a nonsense mutation (A1635C) in the fifth transmembrane domain that produces a truncated receptor, with 
reduced number and affinity of receptors in fransfected cells, and a complete lack of signal transduction when 
exposed to hCG; (2) a missense mutation (Ser616Tyr) that does not alter receptor affinity but markedly impairs 
hCG-induced cAMP production per binding site. Interestingly, the known inactivating mutations are in the same 
region, transmembrane domains 5 and 6, as the activating mutations of FMPP, and they appear to interfere 
primarily with signal transduction. We hypothesize that as larger numbers of these patients are studied there will 
also be inactivating mutations that interfere primarily with hormone binding. 







O 



ZOl HD 00610-15 DEB 

b. Familial male precocious puberty-treatment with combined antiandrogen and aromatase inhibitor 

A long-term pilot study is ongoing to test the hypothesis that the growth rate, bone maturation, and adult height 
of these boys can be normalized with the regimen of antiandrogen (spironolactone) aromatase inhibitor 
(testolactone),and LHRH analog (deslorelin [for those boys who develop secondary LHRH-dependent precocious 
puberty]). We have also initiated a new project to improve this regimen through the use of a more potent 
aromatase inhibitor (CGS 16949A [fadrozole]). 

c. McCune-Albright syndrome - treatment with the aromatase inhibitor testolactone 

A long-term pilot study is ongoing to test the hypothesis that the aromatase inhibitor, testolactone, can improve 
the growth rate, bone maturation, and adult height of girls with this disorder. We have also initiated a new 
project to improve this regimen through the use of a more potent aromatase inhibitor (CGS16949A [fadrozole]). 

d. LHRH analog therapy of central precocious puberty 

Analysis of mean adult height (-1.1 SD) in the first 44 children to attain adult height or near-adult height (within 
2 cm) indicates a highly significant improvement over pretreatment predicted height (-2.0 SD) but a clear shortfall 
compared to expected height based upon parental height (+ 0.1 SD). However, the first children treated had the 
greatest delay of treatment from onset of symptoms (> 3 years), and thus it would be premature to conclude that 
this is the best result that can be achieved. Continued follow up of those children who were treated later and are 
still growing will test our hypothesis that there will be an even greater improvement in the final height of these 
children. 

2. Mechanism and optimization of prepubertal and pubertal growth 

a. The role of estradiol in prepubertal growth 

To test the hypothesis that girls secrete more estrogen than do boys during the prepubertal years, we developed 
an ultrasensitive recombinant cell bioassay for estradiol, based upon yeast cells genetically engineered for extreme 
sensitivity to estrogen, with a sensitivity of 0.02 pg/mL, approximately 100-fold more sensitive than previous 
radioimmunoassays. With this assay, Karen Oerter Klein and Jeffrey Baron showed that estradiol levels in 
prepubertal girls (0.6 ± 0.6 pg/mL) are significantly higher than the level in boys (0.08 ± 0.2 pg/mL). This may 
explain the more rapid bone maturation, the earlier entry into puberty, and the earlier cessation of growth of girls 
compared to boys. 

Recently, we have also used this assay to evaluate estradiol suppression by a new aromatase inhibitor (letrozole) 
in postmenopausal women with breast cancer. Letrozole, at the dose of 100 ug, reduced estradiol fi-om 1.95 to 
0.07 pg/mL, suggesting that this drug should prove useful in the treatment of estogen-dependent conditions. From 
a broader perspective, this study suggests the potential range of applications of this new assay method. Indeed, 
in an editorial in the Journal of Clinical Investigation accompanying this publication. Dr. Jean Wilson stated that 
it should lead to a "renaissance in estrogen physiology." 

b. Saltatory versus continuous model of linear growth 

Traditionally, linear growth has been considered a continuous process, the cummulative result of millions of 
unsynchronized chondrocyte divisions throughout the growth plates in the long bones and spine. Recently, this 
view was challenged in a study that concluded that human growth occurs by a process of saltation (sudden spurts) 
separated by long periods of no growth. These observations, if correct, would require fiindamental revisions in 
our understanding of growth, since none of the known endocrine regulations of growth fluctuate in the manner 
that would be required to synchronize cell divisions throughout the organism. 

We attempted to confirm the hypothesis of saltatory growth by performing 4 different growth measures daily for 
30 days in 5 infants. A method of blinding was developed to prevent observer bias. By 4 different statistical 



0' 



ZOl HD 00610-15 DEB 

approaches, the observed data agreed significantly better with a saltatory than with a continuous model. Thus, 
our data did not support the proposed saltatory model and suggested instead that growth occurs continuously. 

c. Treatment of Turner syndrome 

Based upon the results of our earlier short-term studies, a long-term, randomized, double-blind, placebo-controlled 
clinical trial has been initiated to assess the effect on adult height of estrogen, growth hormone, and combined 
estrogen and growth hormone treatment. The project has been organized as a two-site project, with 1/3 of the 
patients to be followed at Thomas Jefferson University and 2/3 at the NIH, under a joint venture agreement with 
Eli Lilly and Company, which will provide biosynthetic human growth hormone and certain additional support 
for the study. Approximately 140 patients out of a projected total of 160 patients have been enrolled in the study. 

Although the major endpoints of this study remain blinded until the study conclusion, analysis of certain baseline 
and interim data has revealed several new findings. First, analysis of the baseline lipid levels from 137 Turner 
girls and 70 normal girls showed that Turner girls above age 11 have significantly greater cholesterol levels (190 
± 23 vs. 165 ±26 mg/dL) after adjustment for age, karyotype, and body mass index (p<0.01). These increased 
levels precede estrogen or growth hormone treatment. Second, questionnaires from both the parents and the girls 
with Turner syndrome revealed weaker social relationships, school performance, and self-esteem compared to 
control girls matched for age, socioeconomic status, and verbal IQ. Third, cognitive tests of the Turner girls has 
revealed a charactertistic profile of impaired performance on tests of spatial recall, recognition, and reasoning 
(representing in magnitude about 1 SD unit relative to the control population). This cognitive profile appeared 
quite consistent from childhood through adolescence. Future analyses will determine the effect of sex steroid 
administration on this cognitive phenotype, and will examine the cognitive phenotype in girls with unusual 
karyotypes involving partial deletion of one X-chromosome in order to determine the relationship between the 
specific X-chromosomal region deleted and the cognitive phenotype. Thus, the early findings from this trial have 
revealed new metabolic, behavioral, and cognitive features of the Turner syndrome. 

d. The role of growth factors in epiphyseal growth 

An extensive body of knowledge suggests that cell division requires stimulation by growth factors that may be 
endocrine, paracrine, or autocrine in origin. However, the nature, origin, and regulation of the growth factors that 
control cell division in human epiphyseal cartilage remain unknown. The production by recombinant DNA 
methods of factors important for the growth of cuhured cells has made it possible to begin to assess the clinical 
and physiological significance of these growth factors. To examine the potential roles of transforming growth 
factor-P (TGF-P), fibroblast growth factor (FGF), and platelet-derived growth factor in epiphyseal cartilage, we 
have initiated a collaborative project to examine the hormonal regulation of these peptides by immunostaining 
with specific antisera. We will also attempt to examine hormonal regulation of the mRNAs for these peptides 
in epiphyseal cartilage by in situ hybridization and/or solution hybridization. Lastly, we have developed a method 
to infuse these factors, or their antagonists, into the rabbit epiphyseal growth plate, and to measure the subsequent 
growth response. 

Infusion of basic FGF into the rabbit proximal tibial epiphyseal growth plate caused a dramatic induction of 
growth plate cartilage ossification. This observation suggests an important role for basic FGF in the biology of 
longitudinal bone growth, particularly in the terminal differentiation of hypertrophic chondrocytes and their 
invasion and replacement by bone. 

e. Mechanism of glucocorticoid-induced growth failure and subsequent catch-up growth 

Supraphysiologic levels of glucocorticoid cause a profound arrest of epiphyseal growth. This poses a problem 
both in endogenous Cushing's syndrome in children and in children who require supraphysiologic glucocorticoid 
doses as treatment for asthma, juvenile rheumatoid arthritis, and other disorders. 

Previous studies in our laboratory showed that glucocorticoid-induced growth suppression could be induced in 
a single proximal tibial epiphysis by the local infusion of dexamethasone. The mechanism of this local growth 



Ot^ 



ZOl HD 00610-15 DEB 

arrest did not involve suppression of mRNA for growth hormone receptor in the affected epiphysis. Recently, 
we have used this growth suppression model to examine the hypothesis that catch-up growth after glucocorticoid 
withdrawal results from a neuroendocrine mechanism. Contrary to this hypothesis, catch-up growth after 
termination of the dexamathasone infiision was observed only in the epiphysis that had received dexamethasone. 
No change in growth rate was observed in the control contralateral epiphysis, or in the ipsilateral distal tibial 
epiphysis. Thus, catch-up growth following glucocorticoid withdrawal was a local phenomenon that occurred 
only within the affected growth plate. 

f. Mechanism of non-GH-deficient short stature 

We previously published, in the New England Journal of Medicine, a comparison of diagnostic methods for 
growth hormone (GH) deficiency in 54 short children. The study found low sensitivity and diagnostic accuracy 
of the mean spontaneous 24-hour GH level compared to the peak GH level during 3 GH stimulation tests. 

This original study involved only prepubertal children. More recently, we have extended our studies of 
spontaneous GH secretion (now measured as the mean 12-hour nighttime GH level) to include a large cohort of 
short children in each of the 5 stages of puberty. As in the original study of prepubertal short children, we 
observed a very low incidence of abnormally low mean nighttime GH levels among pubertal children with 
idiopathic short stature. Thus, low spontaneous GH secretion is a highly unusual cause of growth failure in 
pubertal, as in prepubertal, short children, and the routine use of spontaneous GH measurement to detect GH 
neurosecretory dysfiinction in such children is unwarranted. 

To test the hypothesis that the diagnostic useftilness of spontaneous growth hormone (GH) measurements could 
be improved by conducting measurements under circumstances that augment GH secretion, we propose to conduct 
these measurements during fasting and after the brief administration of sex steroid. We hypothesize that these 
measures will augment spontaneous GH levels in non-GH-deficient short stature but not in true GH deficiency, 
thus giving rise to a cimically useful test for patients in whom the GH stimulation tests yield equivocal results. 

g. Molecular basis of Laron syndrome 

Previous studies from several laboratories have revealed a number of different mutations that impair hormone 
binding or signal transduction by the GH receptor. Recently, we analyzed the GH receptor (GHR) from a 
geographically isolated Chilean boy with Laron syndrome who was the product of a consanguineous marriage. 
Sequencing of the GHR revealed a novel 2-base pair microdeletion leading to a premature stop codon within the 
extracellular domain, upstream of the critical fransmembrane and intracellular domains. This unusual mutation 
thus expands the known molecular mechanisms that underlie this syndrome of extreme growth failure. This 
observation is part of an ongoing effort to understand the molecular basis for unexplained growth failure in 
children. 

h. Treatment of non-GH-deficient short stature 

1) Growth hormone 

Recent studies from several centers have shown that biosynthetic human growth hormone (hGH) can increase 
short-term growth rate in non-GH-deficient short stature. However, short-term effects are not always sustained, 
and treatment- induced acceleration of pubertal onset could negate short-term gains. Moreover, the increasing off- 
label use of hGH for this purpose has given an important public health significance to the determination of 
whether such use is effective and safe. Thus, to address the long-term outcome of such treatment, a randomized, 
double-blind, placebo-confrolled trial has been designed to determine the effect of biosynthetic human growth 
hormone on the adult height of children with non-GH-deficient short stature. A joint venture agreement with Eli 
Lilly and Company has been established to provide growth hormone and additional support for the study. 
Approximately 56 of the total of 80 subjects have been enrolled in this study. 



OG 



ZOl HD 00610-15 DEB 

Although we remain blinded to the major endpoints of the trial, we have been permitted to examine the hypothesis 
that GH treatment induces hypothyroidism in some children with non-GH-deficient short stature. Based upon 
analysis of the first year's data in 20 subjects, we concluded that GH treatment for 12 months does not produce 
sustained alterations in thyroid function in non-GH-deficient children. 

2) LHRH analog-induced pubertal delay 

The hypothesis that adult height can be increased by prolonging the growth period through delay of puberty is 
supported by our observation of greater adult stature in patients with isolated hypogonadotropic hypogonadism 
(IHH) compared to normal subjects. To determine whether similar gains can be achieved through pharmacologic 
delay of puberty by LHRH analog, a randomized, placebo-controlled trial was initiated that is now fully enrolled 
(50 subjects). The results of this trial will not be known until the subjects have attained adult height. 

3) Insulin-like growth factor- 1 

Recombinant human IGF-1, the growth factor believed to mediate the growth-promoting effects of GH, has 
become available for clinical investigation. We have initiated a protocol to test the hypothesis that this agent will 
stimulate growth both in patients with Laron dwarfism, who are resistant to growth hormone due to an absence 
of fiinctional growth hormone receptor, and in children with non-GH-deficient short stature, who have variable 
responses to growth hormone that in some cases resemble those of Laron dwarfs. 

Since the metabolic effects of growth hormone and lGF-1 on glucose metabolism act in opposing directions, we 
hypothesize that the combination of these agents might have fewer adverse metabolic effects than either agent 
alone. Additionally, evidence for synergistic effects of growth hormone and IGF-1 in several systems in vitro 
suggests the hypothesis that they also may have synergistic effects upon epiphyseal growth in vivo. Thus, we 
envision, once the effects of IGF-1 alone have been determined, additional studies to test the hypothesis of a 
synergistic effect of these agents upon linear growth. 

3. Differential diagnosis of delayed puberty 

No satisfactory diagnostic test is available to determine whether a child with delayed puberty has 
hypogonadotropic hypogonadism and will never enter puberty spontaneously or has constitutional delay of puberty 
and will eventually enter puberty on his own. The recent observation that cholecystokinin can stimulate LHRH 
secretion suggests the hypothesis that children with hypogonadotropic hypogonadism, who lack normally 
flinctioning LHRH neurons, will fail to respond to cholecystokinin, whereas children with constitutional delay 
of puberty will respond normally. 

4. Treatment and molecular mechanism of hypoparathyroidism 

To test the hypothesis that synthetic parathyroid hormone 1-34 (PTH) could maintain normal serum calcium in 
hypoparathyroid patients with reduced urine calcium compared to conventional treatment with calcitriol, Karen 
Winer performed a randomized, cross-over trial comparing these two agents. As we had predicted, PTH permitted 
equivalent control of serum calcium while achieving a significant reduction in urine calcium. Based upon these 
findings, we have extended this pilot study to test the hypothesis that PTH treatment will reduce the incidence 
of renal complications (such as nephrolithiasis, nephrocalcinosis, and decreased renal function) in this disorder. 

In the course of this clinical trial, we received referrals of several families with autosomal dominant 
hypoparathyroidism. A noteworthy feature of these families was that they all had impressive hypercalciuria, even 
when serum calcium was below the normal range. Following the cloning of the calcium-sensing receptor in 
December 1993, we hypothesized, based upon our studies of familial male precocious puberty, that the mechanism 
of this disorder would be an activating mutation of the calcium-sensing receptor. Jeffrey Baron showed this to 
be the case. One family had a mutation in the transmembrane domain region, which presumably activates signal 
transduction. Another family had a mutation in the extracellular domain, where it may act to increase the receptor 
affinity for calcium. Additionally, we found de novo activating mutations of the calcium-sensing receptor in two 



0'. 



ZOl HD 00610-15 DEB 

children with sporadic hypoparathyroidism and marked hypercalciuria. 

The activating mutations of the calcium-sensing receptor predicted a previously unrecognized phenotype of 
marked hypercalciuria in autosomal dominant hypoparathyroidism. Since the calcium-sensing receptor is 
expressed in kidney, where it is believed to negatively regulate calcium reabsorption, an activating mutation of 
this receptor would be expected to cause hypercalciuria even when serum calcium was normal or low. Direct 
comparison of urine calcium in patients with inherited and acquired hypoparathyroidism showed that the patients 
with the autosomal dominantly inherited form had significantly greater urine calcium excretion at any given serum 
calcium level. Moreover, they did not reduce urine calcium as serum calcium decreased, in contrast to the 
patients with acquired hypoparathyroidism. 

Significance to Biomedical Research and the Programs of the Institute: 

Growth and puberty are fundamental developmental processes. Growth failure (stature more than 2 standard 
deviations below the normal height for age) affects approximately 1.5 million American children below the age 
of 18. Severe growth failure (stature more than 3 standard deviations below normal) affects approximately 
100,000 children. Particularly for this severely short group, growth failure may have adverse consequences for 
the individual's psychological, social, educational, and career development. Thus, improved understanding and 
treatment of growth failure would benefit a substantial number of children in this country and throughout the 
world. Additionally, progress in understanding the molecular mechanisms of epiphyseal growth may have 
implications for understanding the fundamental processes of growth and differentiation in other tissues. Finally, 
the production of both human growth hormone and insulin-like growth factor- 1 by recombinant DNA technology 
will continue to cause an increasing empiric use of these and other growth-promoting agents in an attempt to 
increase the adult height of children with growth failure. Thus, the safety and efficacy of growth-promoting 
agents in this setting has become a public health issue that requires controlled clinical trials. 

Precocious puberty occurs in approximately 1 in 5,000 children and often has a profound physical and emotional 
impact on the affected individuals. The above studies have been highly successfiil in controlling central and 
peripheral precocious puberty, disorders for which there has previously been no effective treatment. Progress is 
also being made in understanding the pathophysiology of the McCune-Albright syndrome and familial male 
precocious puberty, and in understanding normal human pubertal physiology. Additionally, children with 
precocious puberty have provided a unique model in which to analyze the influence of hormonal, somatic, and 
social-environmental factors on the behavioral changes of puberty, which are of importance both to the individual 
and to society. 

Hypoparathyroidism is a rare but potentially fatal disorder that may be acquired, through autoimmune destruction 
or inadvertant surgical removal during thyroid or parathyroid surgery, or inherited. Although rare, 
hypoparathyroidism represents a unique opportunity for the scientific study of the role of parathyroid hormone 
in bone and calcium metabolism. The knowledge gained through such study may have implications beyond this 
particular disorder, such as to patients with osteoporosis or other metabolic bone disorders. These other disorders 
represent a major public health problem for which improved understanding of parathyroid hormone effects on 
bone could be of therapeutic importance. 

Proposed Course: 

1. The molecular mechanism of familial male precocious pubertv (FMPP) and Leydig cell hypoplasia 

a. DNA will be obtained fi-om additional affected kindreds. PCR and DNA sequencing of the LH receptor gene 
will be performed to determine which mutations other than those that are currently known can give rise to these 
disorders, since each new mutation provides information with which to construct an increasingly precise model 
of the structural basis for LH receptor activation. 



OB 



ZOl HD 00610-15 DEB 

2. Treatment of peripheral precocious puberty 

a. Children receiving testolactone treatment of McCune-Albright syndrome and combined 
testolactone/spironolactone treatment of familial male precocious puberty will be followed until adult height has 
been achieved. Such follow-up is essential to determine whether the apparent early benefits of treatment will be 
sustained and whether any significant adverse effects will emerge. 

b. Improved therapy for peripheral precocious puberty - the aromatase inhibitor (testolactone) employed in our 
studies was chosen because of its excellent long-term safety record. Testolactone, however, is not as potent an 
aromatase inhibitor as would be desirable. More potent aromatase inhibitors are undergoing clinical development. 
Thus, new patients with FMPP will be randomized to receive fadrozole (CGS 16949 A) or testolactone in a 
crossover design, since fadrozole is approximately 500 times more potent than testolactone. Fadrozole will also 
be used to treat girls with McCune-Albright syndrome who are refractory to testolactone. 

Additionally, our long-range objective is to intercept the pathophysiology of FMPP at the molecular level. 
Improved knowledge of mechanism could lead to therapies directed at preventing gonadal activation, such as gene 
therapy or the development of inverse LH agonists capable of restoring basal fiinction in constitutively activated 
LH receptors, rather than the current strategy of blocking the consequences of gonadal activation. 

3. LHRH analog therapy of central precocious puberty 

The children currently receiving treatment will be seen annually or biannually until age 11 or 12, at which time 
treatment will be discontinued. Thereafter they will be seen annually (annual follow-up will be used for the 
subset of patients who are of greatest research interest because of long duration of treatment and/or proximity to 
attaining adult height) or biannually until final height is achieved. We plan to maintain an up-to-date address 
register that will permit subsequent assessment of reproductive function and normalcy of offspring. 

4. Optimization of prepubertal and pubertal growth 

a. Turner syndrome 

We will continue the long-term, randomized, double-blind, placebo-controlled trial to assess the effect on adult 
height of low-dose estrogen, growth hormone, and the combination of estrogen and growth hormone. 

b. Pubertal delay as a treatment to enhance adult height 

The ongoing randomized, double-blind trial of LHRH analog compared to placebo will be continued. The trial 
is fiilly enrolled (50 subjects), and the children are now being followed until adult height is achieved. 

c. Pharmacologic growth hormone treatment in non-growth hormone-deficient in short stature 

We will continue the long-term, randomized, double-blind, placebo-controlled trial of the effect of growth 
hormone on adult height in children with non-growth hormone-deficient short stature. 

d. The role of growth factors in epiphyseal growth 

We will evaluate whether or not the growth in children with GH insensitivity and with non-GH-deficient short 
stature can be stimulated with recombinant human insulin-like growth factor- 1 (rhIGF-1). 

The potential importance of direct administration of growth factors, compared to administration of growth 
hormone (GH) to stimulate growth factors endogenously, is three-fold. First, it may provide a new approach to 
treatment of patients with Laron dwarfism, who have resistance to GH and do not respond to GH treatment. It 
may also be useful in treating patients with familial deletion of the GH gene, who lack tolerance to GH and 
develop growth-attenuating antibodies to GH during GH therapy. Secondly, we postulate that there may be 



00 



ZOl HD 00610-15 DEB 

negative feedback events that restrain the response to exogenous GH. We hypothesize that rhlGF-l may bypass 
such regulatory events, permitting greater responses than could be achieved through GH administration. 
Additionally, there is evidence in several settings of synergistic interaction between GH and lGF-1. Thirdly, the 
effects of GH and IGF-1 on glucose metabolism are opposite, which suggests the hypothesis that the combination 
of these agents may produce less alteration of glucose homeostasis than either agent alone. 

We will examine hormonal regulation of epiphyseal TGF-P, FGF, platelet-derived growth factor, growth hormone 
receptor, and other factors and their receptors, by the techniques of immunostaining, in situ hybridization, solution 
hybridization, and Northern analysis. These factors will also be examined in vivo by administering them or 
removing them through classical physiologic approaches in rabbits or through the development of appropriate 
transgenic mice. 

5. Improved differential diagnosis of delayed puberty 

Since cholecystokinin has been shown recently to stimulate LHRH release, we will study whether cholecystokinin 
can be used to assess LHRH secretion in man. The test will be used to examine the hypothesis that idiopathic 
hypogonadotropic hypogonadal patients will not respond to cholecystokinin, whereas subjects with constitutional 
delay of puberty will respond. 

6. Treatment of hypoparathyroidism 

We have conducted a pilot study to assess the hypothesis that calcium metabolism in hypoparathyroidism can be 
normalized with single daily subcutaneous injections of synthetic parathyroid hormone. The results of the initial 
phase of the study have confirmed this hypothesis. A short-term, follow-up study is now comparing the safety 
and efficacy of a once-daily versus a twice-daily PTH regimen. Once the optimal regimen is defined, we plan 
to conduct a long-term randomized, parallel study to test the hypothesis that PTH treatment can reduce the 
incidence of renal complications compared to calcitriol treatment. 



Protocols: 




Animal 




92-023 


Laue 


94-006 


Yanovski 


Human 




79-CH-112 


Cutler 



Nutritional regulation of growth and insulin-like growth factor mRNA 
expression in rabbit liver, skeletal muscle, and growth plate 

Effects of retinoic acid on rat epiphyseal growth plate 



Treatment of true precocious puberty with a long-acting luteinizing hormone 
releasing factor analog 

This study tests the hypothesis that the growth and pubertal development of children with LHRH-dependent 
precocious puberty can be normalized by treatment with deslorelin, a long-acting LHRH analog. The study 
employs an open design in which the major outcomes are compared with the subjects' pre-treatment baseline 
values (for example, final adult height is compared with predicted height before treatment and hormone levels 
during treatment are compared with the levels before treatment). The final height data are also being compared 
with the subjects' target height, the height of the normal population, and the height of untreated historical controls 
from the literature. 

The current results indicate that deslorelin treatment reduces sex steroid levels to near prepubertal values, 
decreases growth rate and the rate of bone maturation, and produces final heights that are intermediate between 
the pretreatment predicted height and the children's target height based upon their genetic background. We 






ZOl HD 00610-15 DEB 

conclude that the treatment is effective. Observation is ongoing to determine the adult height of those children 
who were recognized and treated earliest (who may have a better outcome), to assess the children's reproductive 
outcome (and thus the reproductive toxicity, if any), and to suppress secondary LHRH-dependent precocious 
puberty in children who are participating in treatment trials for McCune-Albright syndrome, familial male 
precious puberty, and congenital adrenal hyperplasia (see below). 

82-CH-165 Feuillan Testolactone treatment of girls with LHRH analog-resistant precocious puberty 

due to autonomous, non-neoplastic ovarian estrogen secretion 

This study tests the hypothesis that the growth and development of girls with the McCune-Albright syndrome can 
be normalized by treatment with the aromatase inhibitor, testolactone. The long-term portion of the study 
employs an open design in which the outcome measures are compared with the subjects' pretreatment baseline 
values and the normative values for control subjects. 

The results to date indicate substantial improvement in the majority of subjects, with decreased frequency or 
cessation of menses and decreased rates of growth and bone maturation. Some patients appear to escape from 
the effects of treatment after being effectively controlled for 2 to 3 years. This escape is not attributable to 
secondary central precocious puberty. We conclude that testolactone is a safe and effective treatment for the 
precocious puberty of McCune-Albright syndrome; however, normalization of growth and development have not 
yet been achieved, and more effective therapy is needed. 

83-CH-199 Yanovski A double-blind, randomized, placebo-controlled clinical trial of 

luteinizing hormone releasing hormone analog (LHRH A) in pubertal 
patients with extreme short stature 

This study tests the hypothesis that delay of puberty for 4 years by treatment with the LHRH analog, deslorelin, 
will increase the adult height of children with non-GH-deficient short stature and a normally timed puberty. The 
design is a randomized, double-blinded, placebo-controlled clinical trial. All 50 subjects have been enrolled. 

Final outcome measures are not available because the trial is ongoing. A preliminary, interim analysis early in 
the frial (after the first 16 subjects had completed the 4 years of LHRH analog or placebo) showed a significant 
increase in the predicted height of the LHRH analog-treated subjects by 7.6 cm compared to their own 
pretreatment baseline, and by 10.3 cm compared to the placebo-treated patients. We conclude that pubertal delay 
induced by deslorelin significantly increases predicted adult height in adolescents with short stature and a 
normally timed puberty. Whether deslorelin will increase the final height of these patients cannot be determined 
imtil they have stopped growing. 

91-CH-46 Cutler A randomized, double-blind, placebo-controlled clinical trial of the effects of 

growth hormone therapy on the adult height of non-growth hormone-deficient 
children with short stature 

This trial tests the hypothesis that supplemental GH adminisfration will increase the adult height of children with 
non-GH-deficient short stature, without adverse effects owing to supraphysiologic GH dosage. The fimdamental 
rationale for the study is to protect the public health, since thousands of non-GH-deficient children are currently 
receiving GH treatment before there is convincing evidence of its safety or efficacy in this setting. The design 
is a randomized, placebo-controlled, clinical trial. 56 of the 80 subjects have been enrolled. 

Results of the major endpoints of the study are unavailable because the trial is ongoing. However, the hypothesis 
that GH decreases thyroid function in some subjects with non-GH-deficient short stature has been examined 
through a grouped analysis, by an independent statistician, in a manner which preserved the double-blind study 
design. This analysis showed that supplemental GH, at the dose and frequency employed, did not alter thyroid 
function during 12 months of treatment in these subjects. 

85-CH-16 Cutler Spironolactone and testolactone treatment of boys with familial male 



l4 



ZOl HD 00610-15 DEB 

precocious puberty 

This study tests the hypothesis that the growth and development of boys with familial male precocious puberty 
can be normalized by the combination of an antiandrogen, spironolactone, and an aromatase inhibitor, testolactone. 
The study employs an open design in which the major outcomes are compared with the subjects' pretreatment 
baseline values and, in the case of growth, with the published standards for the normal population. 

The current results, from the initial pilot study, show that the regimen is effective in controlling growth, bone 
maturation, and pubertal development until secondary LHRH-dependent precocious puberty ensues. At that point, 
addition of the LHRH analog, deslorelin, can maintain effective control. However, further study is needed to 
determine the final adult height in the treated patients. 

87-CH-152 Cutler A double-blind, randomized, placebo-controlled trial of the effect of 

biosynthetic growth hormone and/or ethinyl estradiol on adult height in 
patients with Turner syndrome 

This trial tests the hypothesis that supplemental GH treatment, with or without ultra-low, replacement doses of 
ethinyl estradiol, will increase the adult height of girls with Turner syndrome without adverse effects attributable 
to the drug regunen. The design is a 4-arm, randomized, placebo-controlled clinical trial, to adult height. The 
four arms are GH, early (age 5-12) low-dose ethinyl estadiol, GH plus early low-dose ethinyl estradiol, and a 
double placebo. The starting ethinyl estradiol doses from age 5-8 are 25 ng/kg/d and from age 8-12 are 50 
ng/kg/d. However, in recognition of the fact that some girls with Turner syndrome undergo spontaneous puberty, 
and that sensitivity to estrogen varies among subjects, the protocol provides for dosage decreases of 50% 
whenever there is breast development below the age of 12 or bone advancement of 1 year within a 6-month 
period. The dosage can be further decreased by 50% decrements (to 25% or 12.5% of the original dose) if breast 
development or bone age acceleration persist. Thus, the actual estrogen doses during the prepubertal years in 
these subjects are extremely small, and are intended to approximate the prepubertal estrogen secretion rate of 
normal girls. After age 12, all subjects receive the same estrogen regimen beginning at the relatively low dose 
of 100 ng/kg/day from age 12 to 14, and doubling annually thereafter until menses occur. Approximately 140 
of the total 160 subjects have been enrolled. 

Major outcome measures from the trial are unavailable because the trial is ongoing. However, analysis of the 
baseline data from 137 girls with Turner syndrome, before estrogen or growth hormone treatment, revealed 
significantly increased cholesterol levels after age 11 compared to control girls (195 ± 38 vs. 165 ± 26 mg/dL, 
p < 0.01), which remained elevated after adjustment for body mass index. 

This trial, and the Canadian Turner syndrome GH trial, are the only ongoing studies in the world in which the 
effect of GH in Turner syndrome is being compared to an untreated control group. Thus, the trial will play an 
important role in resolving this important clinical issue. 

90-CH-123 Cutler Estrogen effects on cognition in girls with Turner syndrome 

This study tests the hypothesis that the cognitive phenotype of Turner syndrome, namely, decreased performance 
on tests of spatial recognition, reasoning, and recall, is attributable to estrogen deficiency and will therefore be 
correctable, at least in part, by estrogen replacement. The study is a companion protocol to project 87-CH-152 
in the sense that most of its subjects are also taking part in that study. 

Results from this study are not available because of the blinded nature of protocol 87-CH-152. However, the 
baseline, pretreatment data have confirmed the characteristic features of the Turner cognitive phenotype and have 
confirmed its stability over time when compared to an untreated adolescent-aged cohort of Turner girls who are 
not taking part in 87-CH-152. The randomized, placebo-controlled design of this study provides an ideal 
circumstance in which to test the hypothesis of sex steroid effects on cognitive function, since there is greater 
control of other variables than has been possible in previous studies. 

90-CH-179 Nunez CGS 16949A treatment of girls with precocious puberty due to gonadotropin- 



ZOl HD 00610-15 DEB 
independent ovarian estrogen secretion 

This pilot study tests the hypothesis that the new aromatase inhibitor, CGS 16949 A or fadrozole, can control the 
precocious puberty of the McCune-Albright syndrome. The study employs an open design in which the outcome 
measures during 6 months before treatment are compared with those during treatment. Long-term outcomes, such 
as adult height, will be compared with the pretreatment predicted adult height, the target height (determined from 
parental height) and the normal height of the population. 

Current results show incomplete blockade of estrogen production at the maximal current dose 240 ug/kg/day. 
We are currently awaiting FDA approval to evaluate the higher dose of 480 ug/kg/day. If this maneuver is still 
not fiilly effective, we plan to test the effectiveness of even more potent aromatase inhibitors once they are 
avilable for pediatric use. 

91-CH-147 Leschek Dose-response study and placebo-controlled crossover trial of the effects of 

IGF-1 therapy on growth velocity in children with GH insensitivity and in 
children with non-GH-deficient short stature 

This study tests the hypothesis that exogenous IGF-I administration can increase the growth velocity of children 
with GH insensitivity or with other forms of non-GH-deficient short stature. The study employs a double-blind, 
randomized crossover design. Results of the study are not available because the trial is ongoing. 

91-CH-83 Cutler A randomized clinical trial of luteinizing hormone-releasing hormone analog 

in pubertal patients with isolated growth hormone deficiency treated with 
growth hormone 

This study tests the hypothesis that depot leuprolide-induced pubertal delay will increase the adult height of GH- 
treated children with classical GH deficiency. The study employs a randomized, open design - all of the children 
receive GH, and approximately one-half are randomized to receive also the LHRH analog, depot leuprolide. 
Results are not available since the trial is ongoing and few subjects have attained adult height. 

92-CH-Oll Winer Treatment of hypoparathyroidism with synthetic human parathyroid hormone 

1-34 

This study tests the hypothesis that once-daily synthetic parathyroid hormone 1-34 (PTH) will normalize serum 
calcium and reduce the incidence of hypercalciuria, compared to conventional treatment with calcitriol, in patients 
with hypoparathyroidism. The study employs a randomized, open, crossover design lasting 10 weeks for each 
arm for the initial dose-adjustment, feasibility phase. Following this initial phase, a second study is testing the 
hypothesis that twice-daily PTH treatment will normalize calcium metabolism better than once-daily treatment. 
This second study also employs a randomized, open, crossover design lasting 1 weeks for each arm. Lastly, a 
long-term study will test the hypothesis that twice-daily PTH will reduce the incidence of renal complications 
compared to twice-daily calcitriol. This study will employ a randomized, open, parallel design with renal 
complications (decreased glomerular filtration rate, nephrolithiasis, and nephrocalcinosis) as the major outcome 
measures. 

The current results indicate that PTH, administered once-daily, normalizes serum calcium as well as twice-daily 
calcitriol while producing significantly lower urine calcium levels at any given level of serum calcium. The 
preliminary results of twice-daily PTH administration indicate that it normalized calcium metabolism more 
effectively than once-daily administation. We conclude that PTH offers better short-term control of calcium 
metabolism than calcitriol in patients with hypoparathyroidism. The effect of long-term PTH administration will 
require fiirther study. 

93-CH-0054 Cutler The relative effects of androgen, estrogen, and the combination of androgen 

and estrogen on growth rate, GH binding protein, lGF-1, and cognitive 
fiinction in Turner syndrome 



ZOl HD 00610-15 DEB 

This study tests the hypothesis that androgen and estrogen treatment will improve the cognitive function of girls 
with Turner syndrome in tests of spatial recognition, reasoning, and recall. Additionally, we hypothesize that 
androgen treatment will have a greater effect on this aspect of cognitive function than estrogen treatment. Lastly, 
the study tests the hypothesis that androgen and estrogen will have additive effects on growth velocity. 

The study employs a randomized, four-arm, open design, with an enrollment goal of 80 subjects. No results are 
available since the study is still ongoing. 



Publications: 

Baron J, Oerter KE, Colli MJ, Yanovski JA, Novosad JA, Bacher JD, Cutler GB Jr. Catch-up growth: a 
mechanism intrinsic to the growth plate. Endocrinology 1994;135:1367-71. 

Baron J, Oerter KE, Yanovski JA, Novosad JA, Bacher JD, Bolander ME, Cutler GB Jr. Induction of growth 
plate cartilage ossification by basic fibroblast growth factor, Endocrinology 1994;135:1790-93. 

Counts DR, Cutler GB Jr. Growth hormone insensitivity syndrome due to point deletion and frameshift in the 
growth hormone receptor, J Clin Endocrinol Metab 1995;80:1978-81. 

Cutler GB Jr. Precocious puberty. In: Hurst, J.E., ed. Medicine for the Practicing Physician, 4th ed. Philadelphia: 
W.B. Saunders, in press. 

Culter GB Jr. Central puberty in disorders with accelerated peripheral maturaton. In: Plant, T.M. and Lee, P.A., 
eds. The neurobiology of puberty. Bristol, J Endocrinol Ltd., in press. 

Heinrichs C, Counts DR, Munson PJ, Cutler GB Jr, Baron J. Daily growth in infants: evidence against saltation 
and stasis. Science 1995;268:442-45. 

Klein KO, Baron J, Colli M, McDonnell DP, Cutler GB Jr. Estogen levels in childhood determined by a new 
ultrasensitive recombinant cell bioassay, J Clin Invest, 1994;94:2475-80. 

Klein KO, Cutler GB Jr. Puberty. In: Hillier SG, Kitchener H, Nielson JP, eds. Scientific essentials of 
reproductive medicine. London: W.B. Saunders, in press. 

Klein KO, Cutler GB Jr. The hypothalamus and neuroendocrine disorders. In: Berg BO, ed. Principles of Child 
Neurology. New York: McGraw-Hill, in press. 

Klein KO, Demers LM, Santner SJ, Baron J, Cutler GB Jr, Santen RJ. Use of ultrasensitive recombinant cell 
bioassay to measure estrogen levels in women with breast cancer receiving the aromatase inhibitor, letrozole, J 
Clin Endocrinol Metab, in press. 

Laue L, Chan WY, Hsueh AJW, Wu SM, Blomberg L, Cutler GB Jr. Genetic heterogeneity of constitutively 
activating mutations of the luteinizing hormone receptor in familial male-limited precocious puberty, Proc. Natl. 
Acad. Sci. (USA), 1995;92:1906-10. 

Laue L, Wu SM, Kudo M, Hsueh AJW, Cutler GB Jr, Griffin JE, Wilson JD, Brain C, Berry C, Grant DB, Chan 
WY. A nonsense mutation of the human luteinizing hormone receptor gene in Leydig cell hypoplasia. Hum Mol 
Genet 1995;4:1429-33. 

McCauley E, Ross JL, Kushner H, Cutler GB Jr. Self-esteem and behavior in girls with Turner syndrome, Devel 
Behav Pediatr, 1995;16:82-8. 



i'i 



ZOl HD 00610-15 DEB 

Rose SR, Leong GM, Yanovski JA, Blum D, Heavner G, Barnes KM, Chipman JJ, Dichek HL, Jacobsen J, Oerter 
KE, Cutler GB Jr. Thyroid function in non-growth hormone-deficient short children during a placebo-controlled 
double-blind trial of recombinant growth hormone therapy, J Clin Endocrinol Metab, 1995;80:320-24. 

Ross JL, Feuillan P, Long LM, Kowal K, Kushner H, Cutler GB Jr. Lipid abnormalities in Turner syndrome, J 
Pediatr, 1995;126:242-45. 

Ross JL, Reiss A, Freund L, Roeltgen D, Cutler GB Jr. Estrogen effects on cognition and social function in 
Turner syndrome. In: Hibi I. and Takano K, eds. Proceedings of Third International Symposium on Turner 
Syndrome. Amsterdam, excerpta Medica, in press. 

Ross JL, Roeltgen D, Cutler GB Jr. The neurodevelopmental transition between childhood and adolescence in 
girls with Turner syndrome. In: Albertsson-Wikland, K. ed. Turner Syndrome in a life span perspective: research 
and clinical aspects. Amsterdam: Elsevier Press, in press. 

Ross JL, Stefanatos G, Roeltgen D, Kushner H, Cutler GB Jr. Turner syndrome: neurodevelopmental changes 
from childhood through adolescence, Am J Med Gen, 1995;58:74-82. 

Yano K, Saji M, Hidaka A, Kosugi S, Polymeropoulos MH, Okuno A, Kohn LD, Cutler GB Jr. A sporadic case 
of male-limited precocious puberty has the same constitutively activating point mutation in 
lutropin/choriogonadotropin receptor gene as familal case, J Clin Endocrinol Metab, 1994;79:1818-23. 

Yano K, Saji M, Hidaka A, Moriya N, Okuno A, Kohn LD, Cutler GB Jr. A new constitutively activating point 
mutation in the luteinizing hormone/choriogonadotropin receptor gene incases of male-limited precocious puberty, 
J Clin Endocrinol Metab, 1995;80:1162-68. 

Yanovski JA, Cutler GB Jr. The reproductive axis: pubertal activation. In: Adashi EY, Rock JA, and Rosenwaks, 
Z, eds.: Reproductive endocrinology, surgery, and technology. New York, Raven Press, in press. 



lo 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00615-15 DEB 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT 180 characters or less. Title must fit on one line between the borders.) 

Endocrine-Immune Interactions 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) IName, title, laboratory, and institute affiliation) 

P.I. G.P. Chrousos Head SPE, DEB, NICHD 

CM. Bamberger Guest Researcher SPE, DEB, NICHD 



Others: 



M. De Castro 

D. Papanicolaou 
C. Stratakis 

C. Tsigos 

E. Webster 
K. Zachman 



Guest Researcher 
Visiting Associate 
Visiting Associate 
Visiting Associate 
IRTA Fellow 
Bio Lab Technician 



SPE, DEB, NICHD 
SPE, DEB, NICHD 
SPE, DEB, NICHD 
SPE, DEB, NICHD 
SPE, DEB, NICHD 
SPR. DEB. NICHD 



COOPERATING UNITS lif any) 

P.W. Gold, E. Sternberg, Clinical Neuroendocrinology Branch, NIMH; R. Wilder, Arthritis and Rheumatism Branch, 
NIAMS; Gabriel Panayi, University of London, London UK; M.A. Magiakou and G. Mastorakos, Athens University 
Medical School. Athens. Greece: Theoharis Theoharides. Tufts University. Boston. MA. 



LAB/BRANCH 



Developmental Endocrinology Branch 



SECTION 

Section on Pediatric Endocrinology 



INSTITUTE AND LOCATION 

National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

1.8 



PROFESSIONAL: 

1.6 



OTHER: 

0.2 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



E (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The purpose of this project is to increase our understanding of the interactions between the endocrine and immune 
systems in both experimental animals and humans. Several immune system products, such as the inflammatory 
cytokines. Tumor Necrosis Factor-a. lnterleukin-1. and Interleukin-6 activate the hypothalamic-pituitary-adrenai 
(HP A) axis and through it suppress and restrain the inflammatory/immune response. Interleukin-6 is particularly 
potent in humans, stimulating not only ACTH and Cortisol but also arginine-vasopressin (AVP) secretion. Plasma 
interleukin-6 is elevated in glucocorticoid deficiency states and after exercise. Elevations of interleukin 6 in 
infectious, inflammatory, and traumatic states may explain the pathogenesis of the Syndrome of Inappropriate AVP 
Secretion observed in these states. The glucocorticoid antagonist RU 486 potentiated the inflammatory/immune 
response to a standard inflammatory stimulus in intact animals, suggesting that endogenous glucocorticoids exert anti- 
inflammatory/immunosuppressive effects at physiological levels. We recently demonstrated that corticotropin 
releasing hormone (CRH") is produced locally at sites of inflammation and has profound pro-inflammatory effects at 
an autocrine/paracrine level. We have called this "immune" CRH . Glucocorticoids and somatostatin suppress, and 
RU 486 markedly augments local secretion of immune CRH at an inflammatory site. Immune CRH was found in the 
ovary and endometrium, where it may participate in the inflammatory phenomena of ovulation, luteolysis. blastocyst 
implantation, and menstruation. RU 486 allowed the identification of a central nervous system defect in rats prone to 
arthritis. In these animals the glucocorticoid response to stress-mediators is inadequate to restrain the immune system 



following an inflammatory insult. The actual defect is global and located at the level of the hypothalamic CRH 
neuron, which responds poorly to all its known stimulants, including several cytokines, as well as serotonin, 
acetylcholine and norepinephrine. This pathophysiologic mechanism is novel and of relevance to human arthritis and 
other autoimmune diseases. Patients with rheumatoid arthritis have defective pituitary-adrenal axis responses to 
inflammatory stimuli and produce excessive amounts of immune CRH in their inflamed joints. Patients with 
Fibromyalgia have a slight hypofunction of their hypothalamic-pituitary-adrenai axis revealed by CRH testing and 
measurements of urinary free Cortisol excretion. Patients with multiple sclerosis have mild hypercortisolism, which is 
sustained by chronic hypothalamic AVP rather than CRH hypersecretion. The human CRH gene contains estrogen- 
responsive elements in its promoter region providing an explanation for the sexual dimorphism in the incidence of 
autoimmune/inflammatory disease. CRH antagonists may be useful in the treatment of autoimmune/inflammatory 
diseases. We found elevated levels of AVP in rats prone to arthritis and patients with rheumatoid arthritis. AVP 
potentiated the inflammatory response of rats, suggesting that this peptide also participates in inflammation. 



PHS 6040 (Rev. 5/92) 



li> 



ZOl HD 00615-15 DEB 

Project Description: 

We are studying the involvement of interIeukin-6 (IL-6) in the interactions between the inflammatory/immune 
response and the endocrine system and its products. We have shown that IL-6 is one of the most potent stimuli 
of the hypothalamic-pituitary-adrenal (HPA) axis and arginine-vasopressin (AVP) secretion in man. We are 
employing recombinant IL-6 as a provocative stimulus of the corticotropin releasing hormone (CRH) neuron in 
humans with potential alterations in the secretion of this neuropeptide. 

Given to rats, the glucocorticoid antagonist RU 486 enhances the inflammatory response and causes enlargement 
of the thymus and spleen, presumably by antagonizing the immunosuppressive effects of endogenous 
glucocorticoids exerted at physiologic secretion rates. 

One of the potential mechanisms by which RU 486 potentiates inflammation is by stimulation of "immune" CRH 
production at the level of the inflammatory site. We have shown that the latter is a major pro-inflammatory agent 
in vivo and that its gene is responsive to estrogen, suggesting that it may play a major role in the sexual 
dimorphism of the inflammatory/immune response and inflammatory/autoimmune disease. The incidence of the 
latter is much higher in females than males. 

RU 486 allowed the identification of a central nervous system defect in Lewis rats, a strain prone to development 
of autoimmune arthritis and other inflammatory states. In these animals the glucocorticoid response to stress- 
mediators is inadequate to restrain the immune system following an insult. This pathophysiologic mechanism 
is novel and of relevance to human rheumatoid arthritis and other inflammatory/autoimmune diseases. We have 
demonstrated that patients with rheumatoid arthritis have subnormal responses of the hypothalamic-pituitary- 
adrenal axis to inflammatory stimuli, which can explain their unrestrained inflammatory response in the joints 
and other tissues. 

Objectives: 

The objectives of this project are to increase our understanding of the physiologic interactions between the 
endocrine and immune systems and of the impact of alterations in these interactions on the development of human 
disease. The knowledge obtained may assist us with improving the diagnosis and treatment of diseases in which 
these interactions are disturbed. 

Methods Employed: 

Methods consist of glucocorticoid and other receptor assays, and in vivo tests of glucocorticoid action (thymus 
involution, glycogen deposition, growth retardation, suppression of inflammation). Radioimmunoassays and/or 
ELISA assays for measurement of pertinent hormones or cytokines are also used. We have developed a thin layer 
and high performance liquid chromatography (HPLC) method and a radioreceptor assay procedure to purify and 
measure RU 486 in biological fluids. We have also cloned human glucocorticoid receptor mutants/isoforms m 
an expression vector and employ a co-transfection system in which a glucocorticoid-responsive reporter gene is 
used to study the functional properties of these mutants/isoforms. We use in situ hybridization and Northern blots 
to measure expression of glucocorticoid, POMC, AVP and AVP receptor and CRH, CRH receptor and CRH 
binding protein mRNA in the hypothalamus, pituitary, and other tissues. We use standard immunohistochemistry 
and/or extraction-HPLC-radioimmunoassay to demonstrate presence and distribution of CRH and other 
neuropeptides and cytokines in tissues. We also employ a hypothalamic organ culture system for examination 
of CRH and AVP secretion and a dispersed anterior pituicyte culture system for examination of ACTH secretion. 
We employ standard nucleic acid extraction and cloning and sequencing techniques. We have developed our own 
antibodies against CRH and other peptides, as well as against isoforms of the glucocorticoid receptor, for 
performing assays, immunohistochemistry. Western blots, and/or immunoneutralization studies. 



ZOl HD 00615-15 DEB 

Progress: 

Effects of Interleukin-6 in Man 

We have performed dose-response studies with recombinant interleukin-6 in humans by administering the cytokine 
parenterally. Interleukin-6 causes sustained elevations of plasma ACTH, Cortisol and arginine-vasopressin, 
without major side effects, such as hypotension or vascular leak phenomena, as one observed earlier with tumor 
necrosis factor-alpha (TNF-a) and Interleukin-1. We have demonstrated that exercise causes an intensity- 
dependent elevation of plasma IL-6 concentration, which is suppressed by pretreatment of the subjects with 
glucocorticoids. Patients with glucocorticoid deficiency have elevated plasma concentrations of IL-6. We have 
demonstrated by cross-correlation analysis of plasma IL-6, ACTH and Cortisol concentrations in patients with 
early, untreated rheumatoid arthritis, that IL-6 has a circadian rhythm with a morning surge preceding that of 
ACTH and Cortisol. We found the IL-6 drives ACTH and Cortisol in the morning, but is also suppressed by the 
latter at later times. 

Usefulness of the Glucocorticoid Antagonist RU 486 

The availability of RU 486 provided the unique opportunity to study two longstanding questions on the effects 
of glucocorticoids on the immune system and inflammatory response. First, are these effects glucocorticoid 
receptor-mediated? And second, do endogenous glucocorticoids exert an effect on the immune system at 
physiologic secretion rates? In an "aseptic inflammation" rat model, glucocorticoids suppress exudate formation, 
leukocyte diapedesis and prostanoid accumulation. RU 486 was able to antagonize the effect of pharmacologic 
doses of glucocorticoids, suggesting that they are receptor-mediated. In addition, RU 486 given alone caused 
enhancement of the inflammatory response, suggesting that endogenous glucocorticoids at physiological secretion 
rates exert suppressive effects on the inflammatory response in the rat. RU 486 also caused enlargement of the 
thymus and spleen in these animals. These promising observations suggested that a glucocorticoid antagonist 
could be used as an immune enhancer in conditions requiring such enhancement (cancer, immunodeficiency). 

We recently found that human tissues contain an endogenous non-ligand binding glucocorticoid receptor isoform, 
which acts a dominant negative inhibitor of the glucocorticoid receptor. This inhibitor may define the sensitivity 
of the immune system to glucocorticoids. Increased expression might cause glucocorticoid resistance and, hence, 
a hyperactive immune/inflammatory reaction. Decreased expression might cause glucocorticoid hyposensitivity 
and hence, immunosuppression. Whether this isoform is involved in the pathogenesis of 

autoimmune/inflammatory disease is examined under project ZOl HD-00618-14-DEB. 

RU 486 was instrumental in identifying a central nervous system defect in rats prone to arthritis in response to 
antigenic stimulation with streptococcal cell wall polysaccharide (SCW). These rats (Lewis rats) had deficient 
CRH, ACTH and glucocorticoid responses to stressors, including inflammatory stimuli. "Histocompatible" rats 
(Fischer rats), which normally do not develop arthritis in response to SCW, do so when treated with RU 486. 
We defined the CNS defect of Lewis rats in the hypothalamic CRH neuron, which is globally underresponsive 
to the inflammatory cytokines, and the stimulatory neurotransmitters serotonin, acetylcholine and norepinephrine. 
We believe the defect is in the 5' regulatory region of the CRH gene or more likely in one of the related 
transcription factors (see specific section below). 

"Tissue" CRF's vs. "Immune" CRH 

The presence of non-hypothalamic "tissue" CRFs was suggested by Brodish in the early 70's. The concept was 
revived by Woloski et al. in 1985. We have employed a specific antibody to prove both the existence of an 
immunoreactive "tissue" CRH and its importance for the immune/inflammatory reaction in vivo, in the whole 
animal. This is separate from the various humoral mediators of inflammation, such as the inflammatory cytokines 
TNF-a, interleukin-1 and interIeukin-6, or lipid mediators of inflammation, such as several eicosanoids and 
platelet activating factor (PAF), which stimulate CRH neuron function and/or pituitary ACTH secretion in vitro 



u 



ZOl HD 00615-15 DEB 

and/or in vivo . 

A number of recent reports suggested that the interactions between the nervous, endocrine and immune system 
were more extensive than those mentioned above. These recent studies presented evidence that CRH had direct 
effects upon leukocytes. For example, CRH directly stimulated production of proopiomelanocortin (POMC)- 
related peptides and secretion of interleukin -1 and -2, stimulated lymphocyte proliferation, enhanced the 
proliferative response of leukocytes to lectins and increased the expression of the IL-2 receptor on T lymphocytes. 
These reports were complemented by the finding of specific CRH binding sites in various subpopulations of 
leukocytes and, finally, by presence of CRH immunoreactivity and CRH mRNA by in situ hybridization and 
Northern blotting, in resting subpopulations of human leukocytes. All these data suggested strongly that CRH 
might have local direct effects upon the immune/inflammatory system. We attempted to identify the potential 
biological role of these effects by first examining the ability of systemic CRH immunoneutralization to influence 
the size of a quantifiable inflammatory response. As such, we employed the aseptic subcutaneous reaction to 
carrageenin. 

We studied male Sprague-Dawley rats that were injected intraperitoneal ly with neutralizing rabbit anti-CRH 
antisera one hour before subcutaneous injection of carrageenin. Both exudate volume and cell concentrations were 
suppressed by approximately 50-60%, whereas no such suppression was observed in control rats pretreated with 
normal saline, normal rabbit sera or anti-TSH rabbit antisera. The specific suppression of the inflammatory 
response observed after CRH immunoneutralization was clearly opposite to what could be expected from 
abrogation of hypothalamic-hypophyseal portal CRH. The latter should have resulted in relative 
hypoglucocorticoidism and enhancement of the inflammatory response, as is seen in animals pretreated with a 
glucocorticoid receptor antagonist. These findings are instead compatible with the view that local effects of CRH 
are proinflammatory. This concept was complemented by two different sets of data. First, parallel experiments 
with TNF-a neutralizing antisera also showed marked suppression of inflammation, approximately of the same 
magnitude as that observed with anti-CRH antisera. TNF-a is a major auto/paracrine proinflammatory agent, 
which directly, or via IL-1 and/or IL-6, activates the inflammatory response. No additive effect was observed 
between anti-CRH and anti-TNF, a finding compatible with common or shared mechanisms of inflammation 
suppression between these two antisera. Second, direct local administration of anti-CRH antisera into the air 
pouch also decreased the inflammatory reaction. 

Since the above findings suggested that CRH might participate in the inflammatory process in vivo as a local 
stimulatory agent, we examined the site of inflammation for evidence of CRH production. Several approaches 
were taken. First, significantly high amounts of immunoreactive CRH were intracellularly detected in the 
inflamed tissues by specific immunohistochemistry, using an affinity-purified anti-CRH antibody. Second, direct 
measurement of CRH immunoreactive content of extracted inflammatory tissue demonstrated a high quantity of 
CRH immunoreactivity at the range of 400pg/g of wet tissue. High CRH concentrations have also been 
demonstrated in the rat hypothalamus and human placenta. Sizing of the CRH-immunoreactive molecule by 
HPLC revealed a fraction eluting at the location of rat/human CRH 1-41, suggesting that CRH acting in 
inflammatory sites, designated "immune CRH', is similar in chromatographic mobility to that produced by the 
hypothalamus and the human placenta. These findings, taken together, strongly suggest that there is a local 
production of a CRH-like molecule by inflammatory tissues, whose immunoneutralization results in decrement 
of inflammation. The tissue concentrations of this molecule are of sufficiently high concentrations to produce 
biological responses, including POMC-derived peptide secretion and regulation of immune fiinctions. We have 
demonstrated that exogenous glucocorticoid or somatostatin suppress immune CRH secretion in a dose-dependent 
fashion, in parallel to the suppression of the inflammatory/immune response. 

To examine whether immunoreactive CRH produced locally, at the inflammatory site, might have distant, 
endocrine effects, we measured plasma concentrations of IR-CRH in carrageenin- and placebo-treated animals 
through the 7h ensuing carrageenin or placebo administration. The concentrations of plasma CRH were quite low, 
at the range of 8-lOpg/ml (-lO'^M) and similar in the two groups of animals throughout the experiment. These 
levels are markedly lower than those of the rat hypophyseal portal blood and not expected to be bioactive in vivo 



ZOl HD 00615-15 DEB 

or in vitro . Based on these data we suggest that immune CRH is an auto/paracrine, rather than endocrine, 
hormone and that rapid metabolism prevents it from reaching biologically significant circulating concentrations. 
In this sense, it is similar to other autacoids with known major proinflammatory or anti-inflammatory activity, 
of which platelet activating factor (PAF) and prostaglandin E2 are examples. Other paradigms of peripheral CRH 
secretion with primarily auto/paracrine function is its production by chromaffin cells of the sympathetic ganglia 
and adrenal medulla and by Leydig cells of the testis. In the former it appears to potentiate catecholamine 
secretion, while in the latter it seems to be involved in an apparent autocrine inhibitory feedback on testosterone 
production. 

The data from our study suggests that CRH plays a dual, antithetical-albeit counterbalanced role in the regulation 
of the inflammatory/immune response. Indirectly, hypothalamic CRH suppresses this response by activating 
glucocorticoid secretion and causing increased sympathetic outflow, two phenomena leading to 
immunosuppression. Immune CRH, on the other hand, has potent proinflammatory actions which can be 
unmasked by use of neutralizing CRH antisera and, perhaps, peripheral antagonists of this peptide. Does immune 
CRH have any peripheral roles in addition to its proinflammatory ones? For instance, it may be involved in 
antinociception by stimulating a known peripheral analgesic, P-endorphin, or by exerting its own direct 
antinociceptive effects. The latter would be analogous to its recently described antinociceptic effects in the central 
nervous system. How are the effects of immune CRH exerted on the immune system at a molecular level? Its 
major second messenger in the pituitary corticotroph is cyclic AMP(4) which has been primarily shown to 
suppress immune functions when elevated. Would this suggest that immune CRH acts through a different 
receptor or second messenger system? Many questions have been generated from this study and clinical 
applications of immune CRH agonists or antagonists as potentiators or inhibitors of the inflammatory/immune 
response can be envisioned. 

Secretion of immune CRH in inflammatory sites appears to be a generalized phenomenon. We extended our 
observations to several experimental and human states, including retinol-binding protein-induced uveitis in rats 
and mice, and human rheumatoid arthritis and Hashimoto thyroiditis. Immunoneutralization of CRH and TNF-a 
ameliorated uveitis in mice only when given in the early phase of the disease, suggesting that both peptides play 
a major role in the initiation and early propagation of inflammation. Glucocorticoids and somatostatin suppresed 
immune CRH secretion, in accordance with their antiinflammatory effects. Glucocorticoids, in fact, stimulated 
somatostatin secretion within the inflammatory sites and antisomatostatin antibodies inhibited in part the 
antiinflammatory actions of glucocorticoids, suggesting that somatostatin may play the role of an intermediary 
auto-paracrine messenger. 

Immune CRH in Reproductive Tissues 

Since both ovulation and luteolysis and blastocyst implantation and menstruation represent aseptic inflammatory 
phenomena, we considered the ovary and endometrium as potential sites of CRH secretion and action. Indeed 
we have demonstrated secretion of CRH in both the rat and human ovary as well as the presence of specific CRH 
receptors in these gonads. The theca and stroma as well as the corpora lutea contained both. We are currently 
studying the role(s) of CRH in the ovary along with its second messenger system in ovarian cells. We found 
decreased CRH expression in the theca and stroma of human polycystic ovaries. We also found that CRH appears 
to inhibit steroidogenesis by cultured luteinized granulosa cells. This suggests that androgen hypersecretion by 
polycystic ovaries may be related to decreased inhibition by CRH. Recently, we also demonstrated CRH in the 
glandular epithelium of the human endometrium at both phases of the cycle, and within the endometrial stromal 
cells and local macrophages in the luteal phase. 

A Central Nervous Svstem Defect Linked to Autoimmune/Inflammatory Disease 

We have demonstrated that the propensity of Lewis rats to develop severe inflammatory arthritis in response to 
streptococcal cell wall polysaccharide (SCWP) is related to their inability to mount an adequate counter-regulatory 
activation of their HPA axis during the inflammatory response. "Histocompatible" Fischer rats, which normally 



:h 



ZOl HD 00615-15 DEB 

do not develop arthritis in response to SCWP, do so, when treated with the glucocorticoid antagonist RU 486. 
The diminished HPA axis response in Lewis rats appears to result from a generalized defect in the synthesis and 
secretion of hypothalamic CRH in response to different types of physiologic and pharmacologic stimuli. We 
believe that a defect in the CRH neuron of Lewis rats, which makes it globally under-responsive to IL-1, 
serotonin, acetylcholine norepinephrine and other physiologic regulators, is responsible for the hyposecretion of 
glucocorticoids during the immune/inflammatory response. Interestingly, the Sprague-Dawley rat, from which 
the first histocompatible Lewis and Fischer rats were bred, has an intermediate position between the two other 
strains in both HPA axis activity and propensity to develop autoimmune inflammatory disease in response to 
SCWP. We have studied in detail the basal and stress-induced pattern of ACTH and corticosterone secretion in 
the Lewis and Fischer rats. A blunted circadian rhythm, with lowered time-integrated ACTH and corticosterone 
secretion, and hyporesponsiveness to any kind of stress (inflammatory, immobilization, and other) characterizes 
the Lewis rat. We extended the original findings with SCWP to another form of inflammation, the carrageenin 
model - with similar differences between the strains. Interestingly, although the hypothalamic CRH response was 
decreased in Lewis rats compared to Fischer rats, copious amounts of IR-CRH were found in the joints and 
carrageenin inflammation sites of the former. This suggests that both lack of glucocorticoid-induced suppression 
and excess of local CRH secretion in the Lewis rat may be responsible for the excessive inflammatory response 
observed in this strain, and that the regulation of CRH synthesis/secretion is different between the CNS and the 
periphery. The latter could be explained by different tissue-specific regulation or be deficient local 
glucocorticoid-mediated suppression of immune CRH synthesis/secretion. Interestingly, Lewis rats had elevated 
plasma levels of arginine vasopressin (AVP) compared to Fischer rats. Immunoneutralization of AVP ameliorated 
carrageenin-induced inflammation, suggesting that AVP participates in inflammation as a proinflammatory agonist. 
The Lewis and Fischer rats have another fascinating property, when compared to the maternal Sprague-Dawley 
rat strain. The former has behaviors characteristic of low central nervous system (CNS) CRH, whereas the latter 
has behaviors typical of high CNS CRH. These strains represent, thus, interesting models of stress system hypo- 
and hyperfunction, reminiscent of the data described in atypical and melancholic depression, respectively. 

The major question comes up whether the above findings bear any relevance to human pathophysiology. We have 
demonstrated presence of high levels of IR-CRH with the chromatographic mobility ofhCRH 1-41 on HPLC in 
synovial fluid and histologic specimens from joints of patients with rheumatoid arthritis. Severe behavioral 
problems in such patients have been noted, reminiscent of atypical depression, and the question as to whether a 
subgroup of human patients with rheumatoid arthritis or other autoimmune inflammatory diseases have inadequate 
central "endocrine" counter-regulatory responses upon the immune/inflammatory response, combined with 
behavioral traits related to changes in the regulation of the HPA axis, is actively being pursued. We have 
evidence that patients with rheumatoid arthritis are indeed hyporesponsive to inflammatory stimuli, having thus 
pathophysiologic mechanisms similar to those observed in the Lewis rat. We are planning to study this further 
by employing recombinant interleukin-6 (IL-6) as a test stimulus in patients with autoimmune diseases or 
vulnerability to such diseases. 

We studied the HPA axis of patients with fibromyalgia and multiple sclerosis. The former had evidence of 
hypoflinction of this axis, similarly to patients with chronic fatigue syndrome. The latter had mild chronic 
hypercortisolism, which however was sustained by high hypothalamic AVP rather than CRH secretion. We also 
studied the HPA axis of postpartum women, given that this period is characterized by increased incidence of 
autoimmune/inflammatory diseases. We found transiently suppressed hypothalamic CRH secretion in these 
patients, which would explain their increased vulnerability to such diseases (please see project ZOl HD-00618-14- 
DEB). 

Significance to Biomedical Research and the Program of the Institute : 

Interleukin-6 is a very potent and relatively innocuous stimulus of the HPA axis in man. We have shown that 
it carries promise as a diagnostic tool and as a probe to study adrenal physiology. As a challenger of the 
hypothalamic-pituitary-adrenal axis it holds promise in the study of disorders of the axis: Cushing's syndrome, 
adrenal insufficiency, psychiatric hypercortisolism and hypocortisolism and vulnerability to psychiatric and 



9 



ZOl HD 00615-15 DEB 

autoimmune disease. As an experimental tool, RU 486 provides an excellent means to study stress physiology 
and the role of glucocorticoids upon the immune system and the inflammatory response. Recently, RU 486 gave 
us new insight on the pathogenesis of arthritis and/or other inflammatory/autoimmune diseases. In these studies, 
CRH was found to be a major local cytokine, influencing positively the inflammatory response. The CRH gene 
was found to be responsive to estrogen, suggesting that the female-male differences in the inflammatory/immune 
response and the incidence of inflammatory/autoimmune disease might be explained by sexual dimorphism in the 
regulation of this gene. CRH was found to participate in ovarian and endometrial functions, probably promoting 
physiologic aseptic inflammatory processes. Preliminary studies suggest that it may also play a role in blastocyst 
implantation. 

Proposed Course: 

1) We have used interleukin-6 as a new provocative test of hypothalamic-pituitary-adrenal function. We are now 
completing studies with psychiatric hypercortisolism (melancholic depression, pseudoCushing) and 
hypocortisolism (atypical depression, chronic fatigue-fibromyalgia, postpartum blues/depression) and 
autoimmune/inflammatory disease (rheumatoid arthritis, Hashimoto thyroiditis), conditions in which the pituitary 
ACTH reserve or the glucocorticoid negative feedback may be significantly altered. Of special interest would 
be the detection of individuals premorbidly, to define who is vulnerable to develop a disease characterized by a 
hyperresponsive (melancholic depression, panic anxiety) or hyporesponsive (atypical depression, chronic 
fatigue/fibromyalgia, postpartum blues/depression, autoimmune/inflammatory diseases) HPA axis. 

2) We are now defining the anatomic locus and the molecular mechanisms of the defective HPA axis response 
of Lewis rats. It appears that there is generalized deficiency of CRH neuron responsiveness to various stimuli. 
We plan to clone the 5' regulatory region of the CRH gene in these animals, to sequence it and to examine its 
functional activity in the presence of Lewis and Fischer rat hypothalamic nuclear extracts. 

3) It is of utmost importance to define the actual cellular target(s) of immune CRH, to demonstrate the presence 
of CRH receptors in these tissues, to identify the type of receptors (CRH-Rl, -R2a or -R26) present and to show 
whether the recently characterized CRH-responsive element-binding protein is present in such target cells. We 
plan to employ newly developed CRH antagonists to explore the therapeutic potential of these compounds in 
inflammatory states as topical antiinflammatory drugs or as anti-edema/anti-vascular permeability agents. 

4) We plan to examine the potential functional significance of immune CRH in reproductive tissues, using newly 
developed potent CRH antagonists. We have a particular interest in the phenomenon of blastocyst implantation, 
an aseptic inflammation-like process. 

Protocols: 

Human 

82-CH-45 Chrousos Dose-Response Relationships for Single Doses of Corticotropin- 

Releasing Hormone (active) 

This blanket protocol enables us to administer ovine corticotropin- 
releasing hormone (CRH) as a provocative test of the hypothalamic- 
pituitary-adrenal (HPA) axis in men, women, and children with 
potential alterations of this axis. The peptide continues to be an 
investigational drug (rND# 19802) and all data concerning its 
administration are captured and reported annually to the FDA. During 
the past year oCRH was administered under this and other approved 
protocols. 



1-^ ^ 



8-CH-120 



94-CH-134 



Chrousos 



Papanicolaou 



ZOl HD 00615-15 DEB 

The Hypothalamic-Pituitary-Adrenal Axis in Pregnancy, the 
Postpartum Period and in Postpartum Depression Syndromes (precis 
and brief analysis included in project ZOl HD-00618-14-DEB) 

Dose-Response Relationships for Single Doses of Recombinant 
Interleukin-6 in Normal Volunteers and Patients with Disorders of the 
Hypothalamic-Pituitary-Adrenal (HPA) Axis 

This protocol concerns the administration of recombinant interleukin-6 
to normal volunteers and patients with disorders of the HPA axis. 
Interleukin-6 is an investigational drug (INDtf 5419) with which we 
have already obtained some experience from a phase 1 study of 
cancer patients done by NCI. A dose-response curve in normal 
volunteers has been completed and a dose has been selected to be 
administered to the various patient groups listed in the protocol. The 
hypothesis of the protocol is that as a hypothalamic stimulus of CRH, 
acute administration of interIeukin-6 will distinguish biochemically 
not only patients with a clearly disordered HPA axis, i.e. Cushing's 
vs. pseudoCushings's vs^ melancholic depression vs^ chronic active 
alcoholism or melancholic vs^ atypical depression, but also premorbid 
states of vulnerability, i.e. individuals prone to melancholic depression 
or anxiety disorders or individuals prone to atypical depression, 
chronic fatigue/fibromyalgia, postpartum blues or depression, 
alcoholism or autoimmune disorders. 



Animal Protocols: 

89-036 rat Chrousos 



Regulation of Hypothalamic CRH and Pituitary ACTH Release 
(active) 



91-036 rat 



Chrousos 



92-032 rat Webster 



Dose-Response Studies Between Dexamethasone and Suppression of 
Thymic and Adrenal Weights in Fischer and Lewis Rats (completed) 

Reproductive Tissue Inflammation-like Processes and Local CRH 
Production (active) 



95-014 rat 



Chrousos 



Role of Corticotropin Releasing Hormone in Implantation (active) 



Publications: 

Bamberger CM, Bamberger AM, De Castro M, Chrousos GP. Glucocorticoid receptor-beta, a potential 
endogenous inhibitor of glucocorticoid action in humans, J Clin Invest 1995;95:2435-41. 

Chrousos GP. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation, N Engl J Med 
1995;332:1351-62. 

Chrousos GP. Interleukin-6 and steroid therapy in sepsis. [Letter to the Editor], N Engl J Med, in press. 

Chrousos GP. Organization and integration of the endocrine system. In: Sperling M, ed. Pediatric 
endocrinology, Philadelphia: Saunders Co, in press. 



9 '-i 



ZOl HD 00615-15 DEB 

Crofford LJ, Pillemer SR, Kalogeras KT, Cash JM, Michelson D, Kling MA, Sternberg EM, Gold PW, Chrousos 
GP, Wilder RL. Hypothalamic-pituitary-adrenal-axis perturbation in patients with fibromyalgia, Arthr Rheum 
1994;37:1583-92. 

Dom LD, Burgess ES, Dubbert B, Kling M, Gold PW, Chrousos GP. Psychopathology in patients with 
endogenous Gushing syndrome: "atypical" or "melancholic" features, Clin Endocrinol, in press. 

Karalis K, Crofford L, Wilder RL, Chrousos GP. Glucocorticoid and/or glucocorticoid antagonist effects in 
inflammatory disease-susceptible Lewis and inflammatory disease-resistant Fischer rats. Endocrinology 
1995;136:3107-12. 

Karalis K, Mastorakos G, Chrousos GP, Tolls G. Somatostatin analogs suppress the inflammatory reaction in 
vivo, J Clin Invest 1994;93:2000-6. 

Karalis K, Mastorakos G, Sano H, Davis C, Wilder RL, Chrousos GP. Somatostatin may participate in the 
antiinflammatory actions of glucocorticoids. Endocrinology 1995;136:4133-8. 

Kawatito Y, Sano H, Kawata M, Yuri K, Yamamura Y, Kato H, Chrousos GP, Wilder RL, Kondo M. Local 
secretion of corticotropin releasing hormone by enterochromafin cells in human colon, Gastroenterology 
1994;106:859-65. 

Kawatito Y, Sano H, Mukai S, Arai K, Kimura S, Yamamura Y, Kato H, Chrousos GP, Wilder RL, Kondo M. 
Corticotropin releasing hormone in colonic mucosa in patients with ulcerative colitis, GUT, in press. 

Magiakou MA, Chrousos GP. Corticosteroid therapy, nonendocrine disease, and corticosteroid withdrawal. In: 
Bardin W, ed. Current therapy in endocrinology and metabolism. St Louis: Mosby-Year Book Inc, 1994; 120-4. 

Magiakou MA, Chrousos GP. Glucocorticoid therapy and withdrawal. In: Bone RC, ed. Current practice of 
medicine, Philadelphia: Curtent Medicine Inc, in press. 

Margioris A, Gravanis A, Chrousos GP. Glucocorticoids and mineralocorticoids. In: Brody T, Lamer J, 
Minneman K, Neu HC, eds. Human pharmacology: molecular to clinical. St. Louis: Mosby- Yearbook, 1994;473- 
81. 

Mastorakos G, Bouzas E, Caspi R, Friedman T, Chrousos GP. Immune corticotropin releasing hormone (CRH) 
is present in the eyes of and promotes experimental autoimmune uveoretinitis (EAU) in rodents. Endocrinology 
in press. 

Mastorakos G, Cizza G, Kvetnansky R, Bergamini E, Blackman MR, Gold PW, Chrousos GP. Aging and acute 
stress decrease corticotropin releasing hormone in the ovary of the Fischer 344/N rat, Life Sci 1995;56:1065-71. 

Mastorakos G, Patchev VK, Chader GJ, Chrousos GP, Gaudet SJ. Differences in arylakylamine N- 
acetyltransferase activity between inflammatory disease-susceptible Lewis and -resistant Fischer rats, 
Neuroendocrinology 1995;62:71-8. 

Mastorakos G, Scopa C, Friedman T, Vryonidou A, Kattis D, Phenekos K, Chrousos GP. Presence of 
immunoreactive corticotropin releasing hormone in normal and polycystic human ovaries, J Clin Endocrinol 
Metab 1994;79:934-9. 

Mastorakos G, Weber JS, Magiakou MA, Gunn H, Chrousos GP. Hypothalamic-pituitary-adrenal axis activation 
and stimulation of systemic vasopressin secretion by recombinant interleukin-6 in humans: potential implications 
for the syndrome of inappropriate vasopressin secretion, J Clin Endocrinol Metab 1994;79:1191-7. 



2^i 



ZOl HD 00615-15 DEB 

Michelson D, Stone L, Galiiven E, Magiakou MA, Chrousos GP, Sternberg EM, Gold PW. Multiple sclerosis 
is associated with alterations inhypothalamic-pituitary-adrenal function, J Clin Endocrinol Metab 1994;79:848-53. 

Papanicolaou D, Chrousos GP. Interactions of the endocrine and immune systems in children and young adults, 
Curr Opin Peds 1995;7:440-4. 

Reincke M, Allolio B, Arlt W, Heppner C, Petcke F, Mbulamberi D, Siekman L, Vollmer D, Winkleman W, 
Chrousos GP. Impairment of adrenocortical function associated with increased plasma tumor necrosis factor-alpha 
and interleukin-6 concentrations in african trypanosomiasis, NeuroImmunoModulation 1994;1:14-22. 

Scopa CD, Mastorakos M, Friedman TC, Melachrinou MC, Merino MJ, Chrousos GP. Presence of 
immunoreactive corticotropin releasing hormone (CRH) in thyroid lesions, Am J Path 1994;145:1159-67. 

Stratakis CA, Chrousos GP. Capillaritis (purpura simplex) associated with use of aminoglutethimide in Cushing's 
syndrome, Am J Hosp Pharm 1994;51:2589-91. 

Stratakis CA, Chrousos GP. Neuroendocrinology of stress and the immune response. In: Root A, ed. 
Proceedings of the 1994 NCOS investigators meeting. Califon: Gardiner-Caldwell, 1995;4-7. 

Stratakis CA, Mastorakos G, Chrousos GP. Interleukin-6 elevation in critically ill infants with sepsis and 
necrotizing enterocolitis. [Letter to the Editor]. J Peds 1994;125:504. 

Stratakis CA, Mitsiadis NS, Sun D, Chrousos GP, O'Connell A. Recurring oral giant cell lesion in X linked 
hypophosphatemic rickets: clinical manifestation of occult-hyperparathyroidism, J Peds, in press. 

Theoharides TC, Spanos C, Pang Z, Alferes L, Ligris K, Letoumeau R, Rozniecki JJ, Webster E, Chrousos GP. 
Stress-induced intracranial mast ceil degranulation. A corticotropin releasing hormone-mediated effect. 
Endocrinology, in press. 

Tsigos C, Chrousos GP. The neuroendocrinology of stress. In: Krotowski D, Nosek M, Turk M, eds. Women, 
health and disability. Baltimore: Brookes Publishers, in press. 

Tsigos C, Chrousos GP. Physiology of the hypothalamic-pituitary-adrenal axis in health and dysregulation in 
psychiatric and autoimmune disorders, Endocrin Metab Clin N Amer 1994;23:451-66. 

Tsigos C, Chrousos GP. Stress, endocrine manifestations and diseases. In: Cooper CL, ed. Handbook of Stress 
Medicine. Boca Raton, FL: CRC Press, in press. 

Vamvakopoulos NC, Chrousos GP. Hormonal regulation of human corticotropin releasing hormone gene 
expression: implications for the stress response and immune/inflammatory reaction. Endocrine Reviews 
1994;15:409-20. 






DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00616-14 DEB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT 180 characters or /ess. Title must fit on one line between the borders.) 

Structure, Function, and Physiology of Glycoprotein Hormones 



PRINCIPAL INVESTIGATOR /List other professional personnel below the Principal Investigator.) IName, title, laboratory, and institute affiliation) 

P.I. M.R. Flack Senior Clinical Investigator SME, DEB, NICHD 



Others: 



B.C. Nisula 
D. Blithe 
J. Anasti 
J. Froehlich 
C. Finch 



Head 

Expert 

Clinical Associate 

Visiting Fellow 

Biotechnician 



SME, DEB, NICHD 
SME, DEB, NICHD 
SME, DEB, NICHD 
SME, DEB, NICHD 
SME, DEB, NICHD 



COOPERATING UNITS (if any) 

None 



LAB/BRANCH 

Developmental Endocrinology Branch 



SECTION 

Medical Endocrinology Section 



INSTITUTE AND LOCATION 

National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

4.0 



PROFESSIONAL: 

3.5 



OTHER: 

0.5 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects H (b) Human tissues 
D (a1) Minors 
D (a2) Interviews 



n (c) Neither 



SUMMARY OF WORK {Use standard unreduced type. Do not exceed the space provided.) 

This project has been terminated. 



PHS 6040 (Rev. 5/92) 



21) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00618-14 DEB 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Physiology and Pathophysiology of the Hypothalamic-Pituitary-Adrenal and Gonadal Axes 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: G.P. Chrousos Head SPE, DEB, NICHD 



Others: 



See attached list. 



COOPERATING UNITS (if any) 

See attached list. 



LAB/BRANCH 

Developmental Endocrinology Branch 



SECTION 



Section on Pediatric Endocrinology 



INSTITUTE AND LOCATION 

National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



PROFESSIONAL: 



6.6 



OTHER: 



1.4 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects E (b) Human tissues 
D (a1) Minors 
D (a2) Interviews 



D (c) Neitiier 



SUMMARY OF WORK (tJse standard unreduced type. Do not exceed the space provided.) 

In this project we seek to advance the understanding of the phvsiology and pathophvsiologv of the hvpothalamic- 
pituitary-adrenal (HPA) and -gonadal (HPG) axes . The role of stress-related hormones in normal and disease states is 
being examined, and clinical applications for these hormones are sought. The discovery of the structure of corticotropin 
releasing hormone (CRH) and the development of sensitive assays for measuring HPA- and HPG-related hormones and 
their receptors have led to rapid progress in this field. Major progress has been made in three areas: 1) Clinical 
applications of CRH: An ovine (o) CRH stimulation test has been developed that is useful in the differential diagnosis 
of adrenal insufficiency. Gushing 's svndrome , and pseudo-Cushing's states (psvchiatric hvpercortisolism) . The oCRH 
test and/or measurements of CSF CRH have increased our understanding of the pathophysiology of Cushing's 
syndrome, melancholic depression , childhood sexual abuse , atypical/seasonal depression , the chronic 
fatigue/fibromvalgia svndromes , diabetes mellitus , rheumatoid arthritis , and the postpartum blues/depression svndromes . 
2) The regulation of the axis by neurotransmitters, neuropeptides, and glucocorticoids has been studied in vivo and/or in 
vitro . Third trimester pregnant women and athletes have a hyperfiinctional pituitary-adrenal axis in the resting state. 
The hCRH gene 5' regulatory region has been cloned and sequenced and its regulation has been studied. It has 2 active 
promoters and responds positively to estrogens, providing a potential explanation for the sexual dimorphism of 
psychiatric diseases characterized by aberrations in CRH secretion. 3) Roles and actions of HPA and HPG axes 
hormones. Glucocorticoid resistance is an autosomal recessive or dominant disease associated with abnormalities of the 
glucocorticoid receptor . We have elucidated the molecular pathophysiology of this syndrome by defining mutations 
and/or deletion of the glucocorticoid receptor gene leading to abnormal or decreased receptors in the tissues of patients. 
The mineralocorticoid receptor and the subunits of the amiloride-sensitive sodium channel are studied in sporadic cases 
of pseudohvpoaldosteronism or mineralocorticoid resistance . The interaction of the classic glucocorticoid receptor 
(GRa) and its nonligand binding natural homolog glucocorticoid receptor 6 (GR6) with each other and with the heat- 
shock proteins and glucocorticoid-responsive elements (GREs) of the DNA are studied, as the well as the importance of 
GR6 in human physiology and pathophysiology. We have elucidated the molecular pathophysiology of hereditary 
ACTH resistance, an autosomal recessive disorder characterized by isolated glucocorticoid deficiency, by defining 
abnormalities of the ACTH receptor gene. We have elucidated the molecular pathophysiology of testicular and ovarian 
resistance to luteinizing hormone (LH) by defining abnormalities of the LH receptor gene. We have localized the gene 
for Camev Complex , a multiple neoplasia/lentiginosis syndrome, on chromosome 2pl6. 



PHS 6040 (Rev. 5/92) 



Professional Personnel: (continued) 



ZOl HD 00618-14 DEB 



Others: 



K. Arai 

C. Bamberger 
G. Cizza 

M. De Castro 
T. Kino 
M. Karl 
A.C. Latronico 

D. Papanicolaou 
M. Ramayya 

C. Stratakis 
S. Taymans 
C. Tsigos 

E. Webster 
K. Zachman 
S. Yanovski 



Visiting Fellow 


SPE, 


DEB, 


NICHD 


Guest Researcher 


SPE, 


DEB, 


NICHD 


Visiting Associate 


SPE, 


DEB, 


NICHD 


Guest Researcher 


SPE, 


DEB, 


NICHD 


Visiting Fellow 


SPE, 


DEB, 


NICHD 


IRTA Fellow 


SPE, 


DEB, 


NICHD 


Guest Researcher 


SPE, 


DEB, 


NICHD 


Visiting Associate 


SPE, 


DEB, 


NICHD 


Clinical Associate 


SPE, 


DEB, 


NICHD 


Visiting Associate 


SPE, 


DEB, 


NICHD 


Predoctoral IRTA 


SPE, 


DEB, 


NICHD 


Visiting Associate 


SPE, 


DEB, 


NICHD 


IRTA Fellow 


SPE, 


DEB, 


NICHD 


Bio Lab Technician 


SPE, 


DEB, 


NICHD 


Special Volunteer 


SPE, 


DEB, 


NICHD 



Cooperating Units: 

T. Friedman, Laboratory of Developmental Neurobiology, NICHD; P.W. Gold, Clinical Neuroendocrinology Branch, 
NIMH; E. Oldfield, Surgical Neurology Branch, NINDS; P. Deuster, Human Performance Laboratory, Dept of Military 
Medicine, USUHS; P. Peeke, Office of Alternative Medicine, NIH; A. Magiakou, G. Mastorakos, Athens University 
Medical School, Greece; M. Reincke, University of Wurtzburg, Germany; A. Vgontzas, Pennsylvania State University, 
Hershey, PA; D. Fraker, Surgery Branch, NCI; L. Dorn, University of Pennsylvania, Pittsburg, PA; A.J. Carney, Mayo 
Clinic, Rochester, MA; Mark Hughes, Human Genome Institute, NIH; D.Y. Leung, University of Colorado Health Sciences 
Center, Denver, CO. 






ZOl HD 00618-14 DEB 

Project Description 

Objectives: 

The term stress encompasses the sum of body reactions that take place during the influence of stimuli (stressors) 
threatening to alter homeostasis. The changes occurring are both behavioral and physical. Stress is associated 
with activation of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic system. It appears that these 
two systems interact at various levels, from the CNS to the peripheral target tissue, and when activated have 
profound effects on other systems, including those subserving reproduction, growth and immunity. The structure 
of CRH, the principal hypothalamic hormone regulating pituitary-adrenal function, was discovered in 1981 after 
over 20 years of investigation. This discovery made it possible to begin to explore the role of this hormone in 
stress and in human health and disease. The general objectives of this project have been to understand the 
physiologic and pathophysiologic mechanisms of stress in experimental animals and man and to study the fiinction 
of the hypothalamic-pituitary-adrenal axis in stress, health and disease. For clarity, we have divided the 
"Progress", "Significance to Biomedical Research and the Program of the Institute" and "Proposed Course" 
sections of the annual report into three broad sections: a) Clinical Applications and Implications of CRH, b) 
Regulation of the Hypothalamic-Pituitary-Adrenal Axis In Vivo and In Vitro, c) Roles, Actions and 
Pathophysiology of HPA and Hypothalamic-Pituitary-Gonadal (HPG) Axes Hormones, and d) Mechanisms of 
Adrenocortical Tumorigenesis. 

Methods Employed: 

1) Metabolic clearance studies - Methods consist of (1) radioactive labeling and chromatographic purification of 
a hormone or (2) measurement ofthat hormone by radioimmunoassay, immunoradiometric assay or enzyme-linked 
assay and estimation of its metabolic clearance rate by the pulse injection or continuous infusion methods. 

2) Radioimmunoassay (RIA)/Immunohistochemistry - RIAs have been developed to measure ovine and human 
CRH, ACTH and POMC-derived peptides such as beta-endorphin and alpha-MSH, as well as other peptides. 
Also, RIAs have been developed for vasopressin, dynorphin A, tumor necrosis factor-a (TNF-a ), 5-sleep- 
inducing peptide and neuropeptide Y. Affinity purification of antibody and immunohistochemistry procedures 
for several of the above peptides have been set up. 

3) Peptide hormone receptor assays have been used to examine the presence and characteristics of these receptors 
in various tissues or cells. 

4) An ACTH bioassay in which rat adrenal corticosterone secretion is examined as an end-point. 

5) A pituitary culture system is being used to examine the CRH activity of various extrahypothalamic substances 
with CRF bioactivity but not immunoreactivity. 

6) A hypothalamic organ culture system in which the secretion of CRH from rat hypothalami is examined in vitro . 
This allows us to directly examine the regulation of CRH neuron function by neurotransmitters, neuromodulators, 
and feedback systems. 

7) An organ perfusion system of human or rat ftill thickness placenta fragments has been established in which 
we study the secretion of CRH and POMC-derived peptides. The regulation of placental secretion of these 
peptides is studied. 

8) POMC, CRH, AVP mRNA assays by Northern blotting, RNAse protection, and in situ hybridization are used 
to determine the site of POMC, CRH, and AVP gene expression as well as their regulation. Similar methods have 
been established for the CRH and AVP receptors and the CRH-binding protein. 



9 M 



ZOl HD 00618-14 DEB 

9) Glucocorticoid receptor assays - both binding assays, protein assays by Western blotting and assays of receptor 
mRNA by Northern blotting or in situ hybridization for both types of the glucocorticoid receptor, glucocorticoid 
and mineralocorticoid, are performed in our laboratory. The regulation of the glucocorticoid receptor gene 
expression, especially its alternative splicing to the classic glucocorticoid receptor and its natural nonligand- 
binding isoform R, is examined. The molecular pathophysiology of glucocorticoid and mineralocorticoid 
resistance in man and nonhuman New World primates is examined. 

10) Standard cloning and sequencing procedures. These are applied in the cloning of the glucocorticoid, 
mineralocorticoid, ACTH and LH receptor in families or subjects with Cortisol , aldosterone, ACTH and LH 
resistance, and in the cloning of other genes of choice. Co-transfection assays and use of reporter plasmids 
expressing chloramphenicol acetyl transferase (CAT) or luciferase coupled to appropriate promoters have been 
developed to assess the functional activity of several gene products. 

11) Standard genetic linkage analyses for chromosomal localization of hereditary endocrine disorders, such as 
Carney complex and the Triple A syndrome. 

Progress: 

A. Clinical Applications and Implications of CRH 

From testing normal subjects it has become evident that age plays no major role in the responsiveness of plasma 
ACTH and Cortisol to CRH in man. Children above five to old men in their nineties have virtually 
indistinguishable responses. There is a small difference in the responsiveness of the axis between men and 
women characterized by exaggerated ACTH responses and slight prolongation of the Cortisol responses in the 
latter. There are no changes in the responsiveness of the HPA axis during the different phases of the menstrual 
cycle in normal women. 

CRH causes elevations of plasma ACTH and Cortisol in patients with pituitary Cushing's disease, whereas it does 
not in patients with the ectopic ACTH syndrome. The differential diagnostic accuracy of the CRH test in this 
condition exceeds 85 percent. Patients with Cushing's syndrome of adrenal etiology have low or undetectable 
levels of plasma ACTH, that fail to respond to exogenous CRH. Thus, the CRH stimulation test is a useful out- 
patient, brief, safe and easy diagnostic tool in the differential diagnosis of Cushing's syndrome, as it distinguishes 
pituitary from adrenal or ectopic causes, each requiring a different therapeutic approach. A prospective study of 
our patient series suggests that the oCRH test is equal, or superior, in its predictive value than the standard tests 
which are in-patient, long, and cumbersome. 

Pituitary Cushing's disease is usually due to small ACTH-secreting adenomas. Cure is achieved by 
transsphenoidal adenomectomy. We have improved the cure rate by introducing a new sampling method in 
association with oCRH administration. We are sampling the veins draining the pituitary gland (petrosal sinuses) 
simultaneously for measurement of ACTH both basally and after CRH. In the overwhelming majority of patients 
an ACTH concentration gradient is found between one or both petrosal sinuses and a peripheral vein. Thus, the 
surgeon searches for a pituitary adenoma. Continuous ovine CRH administration as early as a week after 
successful, curative surgery stimulates ACTH and Cortisol levels into the normal range, suggesting that the 
profound adrenal insufficiency normally observed in cured patients is of hypothalamic or suprahypothalamic 
origin. 

In adrenal insufficiency, testing with CRH helps differentiate between hypothalamic and pituitary causes, since 
in the former there are ACTH responses to CRH, whereas in the latter there are not. It is interesting that hCRH, 
which we have shown to be short-acting and reproducing the spontaneous secretory episodes of ACTH and 
Cortisol, restores, when given in a pulsatile fashion, the normal pattern of ACTH and Cortisol in patients with 
hypothalamic adrenal insufficiency. Glucocorticoid therapy administered on alternate days is associated with a 
normal ACTH and Cortisol response to CRH on the day off Interestingly, adrenal androgen responses are 



GO 



ZOl HD 00618-14 DEB 

suppressed suggesting that alternate therapy may be a good therapeutic option for growing children with 
hyperandrogenism. Patients with the late-onset form of congenital adrenal hyperplasia had an extensive evaluation 
of the HPA axis. Interestingly, it appeared that the equilibrium between ACTH and Cortisol in these patients as 
a group was totally normal. We considered this an inherent long-standing adaptation of the HPA axis in these 
patients. 

Patients with psychiatric disorders associated with hypercortisolism (depression, panic anxiety, anorexia nervosa) 
have decreased plasma ACTH responses to exogenous CRH. The blunting of the response is inversely 
proportional to the degree of hypercortisolism. Thus, patients with pseudo-Cushing's states could potentially be 
differentiated from patients with Cushing's disease who respond differently to exogenous CRH. The diagnostic 
accuracy of the test is approximately 75% in distinguishing these two conditions. Pretreatment of the patients 
with dexamethasone improves significantly the distinction between the two conditions. 

Recently, the role of CRH has been expanded from merely that of an ACTH secretagogue to that of a 
neuromodulator of broader significance. Intracerebroventricular administration of CRH in rats causes 
physiological (HPA axis activation, sympathetic activation, hypogonadism) and behavioral (aggression, 
irritability, anorexia, loss of libido) changes that are normally observed in stress. These findings have suggested 
a pathophysiologic role of CRH in psychiatric conditions characterized by chronic hypercortisolism. If this 
hypothesis is correct, treatments directed towards preventing endogenous CRH hypersecretion or CRH antagonists 
may be developed for these conditions. 

We compared the diagnostic utility of hCRH to that of oCRH in Cushing's syndrome and psychiatric 
hypercortisolism. The ovine CRH analog is clearly superior to the human one for diagnostic testing. However, 
the hCRH analog will remain useful in studies requiring restoration of the physiologic pulsatile pattern of ACTH 
and Cortisol. 

We have immunized rabbits with CRH and have produced antisera against both ovine and human CRH. We have 
used these antisera to produce immunoneutralization in vivo or to develop radioimmunoassays for these peptides. 
These radioimmunoassays are sensitive to the pg/ml level and have been extensively used to estimate the 
metabolic clearance rates of these peptides in normal men and patients with adrenal insufficiency, Cushing's 
syndrome, depression, and anorexia nervosa. Strong stress stimuli, such as surgery, exercise, or insulin-induced 
hypoglycemia, fail to cause major elevations of plasma CRH immunoreactivity. CRH immunoreactivity can be 
readily measured in the cerebrospinal fluid (CSF). Its concentration is elevated in depression and anorexia 
nervosa and decreased in Cushing's syndrome and Alzheimer's disease, and atypical depression. Interestingly, 
ACTH immunoreactive concentrations in the CSF follow the direction of CRH in the above conditions, suggesting 
that arcuate nucleus ACTH rather than pituitary ACTH is detected in the CSF. 

We examined patients with atypical, seasonal depression. These patients develop dysthymia in the winter months, 
gain weight and are complaining of fatigue and somnolence. In these patients ovine CRH testing in the summer 
was totally normal, whereas in the winter it was indicative of secondary hypocortisolism. Interestingly, light 
therapy normalized the CRH response. We obtained similar data suggesting HPA axis hypofunction in patients 
with the chronic fatigue syndrome, a condition characterized primarily by fatigue, as well as in patients with 
fibromyalgia. These findings suggest that a spectrum of diseases exist, in which the pathophysiology is 
characterized by hypofunction of the central stress system. These syndromes contrast to melancholic depression, 
panic anxiety and anorexia nervosa, in which we have hyperactivity of the CRH system. 

A state-independent abnormally functioning stress system (hyper- or hypo-functioning) might be either a result 
of different heredity or because an environmental stimulus was exerted at some critical period of life, usually 
during development (antenatally, infancy, childhood). A state-dependent abnormally functioning stress system, 
on the other hand, should revert completely to normal once the organism returns to a normal nonpathologic state. 
Unipolar melancholic depression or atypical depression have alterations of the HPA axis on opposite ends of a 
spectrum, which could be hereditary and/or constitutional (i.e. a trait) or state-dependent. Evidence from 



O. 



ZOl HD 00618-14 DEB 

experimental animals and humans accumulates, which indicates that the former is most likely the case. We 
examined several groups of patients and normals with the purpose of understanding better the relation between 
personality, stress, depression, and the HPA axis. These were sexually abused girls with dysthymia several years 
after the abuse took place, children of one or two parents with depression and adolescents with depression, as well 
as appropriate controls for each of the groups. All of these patients had standardized psychological testing, an 
oCRH test and measurement of 24h urinary free Cortisol excretion. Sexually abused girls had a blunted ACTH 
response, a normal Cortisol response and normal urinary free Cortisol excretion. Our interpretation was that these 
patients had constitutional hypersecretion of CRH but normal negative feedback controls that allowed 
normalization of free Cortisol. Children of one or two parents with depression had normal oCRH testing and 
urinary Cortisol excretion. Adolescents with depression also had normal oCRH testing and urinary Cortisol 
excretion. In the latter two groups we have the following potential explanations: (1) they have normal CRH 
secretion and the locus of depression is outside the stress system; (2) they are a mixed group of low and high 
CRH secretors and the averaging is normal; we saw no bimodal distribution; (3) they are low secretors primarily, 
and our means of examination failed to detect their abnormality; indeed, the age of onset for atypical depression 
is adolescence and for melancholic depression the third decade of life. 

We have found that African Americans have higher plasma ACTH levels than Caucasians in response to CRH. 
Further study demonstrated that this is due to different metabolism of ACTH 1-39 in blacks vs. whites, with 
identical ACTH bioactivity, however, in both races. 

We have cloned and sequenced the 5' regulatory region of the human CRH gene and identified recognizable 
signal sequences. The functions of some of these sequences were studied. This gene has 2 different promoters 
with potentially different regulatory sensitivities, and, in addition, 4 half perfect estrogen-responsive elements that 
respond positively to estradiol in an estrogen receptor-mediated fashion. The latter suggests that the sexual 
dimorphism of psychiatric diseases characterized by altered CRH secretion in humans, such as depression and 
anorexia nervosa, might be explained by the differences in the regulation of this gene. 

We hypothesized that marmosets of the species Callithrix jacchus might represent a potentially interesting model 
for human depression. These New World primates maintain a monogamous breeding system with the males and 
older siblings participating in the rearing of infants. Subordinate males or females or offspring in the family are 
reproductively suppressed. Disruption of societal bonds, such as separation of the infant from its parents leads 
to significant behavioral and physical changes. These include in adults, withdrawn behavior, decreased feeding, 
loss of weight, and death ("wasting syndrome") and in infants delay or complete cessation of growth and 
abnormal behaviors. We have documented profound changes in hormones of the HPA axis with degree of 
subordination, and the wasting syndrome and have shown defective growth of infants abused by immature parents. 
The growth retarded infants catch up length-wise and improve behaviorally at later stages of their development. 

B. Regulation of the Hypothalamic-pituitary-adrenal (HPA) Axis In Vivo and In Vitro . 

It has become clear from our studies that opioids are involved in the regulation of the human hypothalamic- 
pituitary-adrenal axis. Morphine both suppresses the CRH-secreting neuron and/or stimulates some corticotroph 
suppressing factor. The overall effect of opioids is to suppress the hypothalamic-pituitary-adrenal axis. It has 
become known from other laboratories that catecholamines, vasopressin and oxytocin stimulate the corticotroph 
in vitro . Vasopressin also shows synergy with CRH in vivo when the peptides are administered together. 

The possibility of peripheral extrahypothalamic CRTs or CRH CRFs participating in the regulation of the HPA 
axis and playing, in addition, other roles in homeostasis during stress, is a lead that we have followed. CRH at 
pharmacologic doses causes marked peripheral vasodilation in primates with a concomitant decrease in blood 
pressure. The major vessels involved were the mesenteric artery and to a lesser extent the iliac arteries. From 
a survey of peripheral tissues for CRH receptors we have found such receptors in the adrenal medulla and 
sympathetic ganglia. These receptors are coupled to cyclic AMP and to catecholamine and enkephalin secretion. 
Receptors on blood vessels or perivascular mast cells may also be present. We are actively searching for 



3^ 



ZOl HD 00618-14 DEB 

peripheral extrahypothalamic (tissue) corticotropin-releasing or CRH releasing-factors. These factors appear to 
have different immunoreactivity from that of hypothalamic CRH. Immune system products, such as interleukins 
1 and 6, thymosin and TNF-a, or tissue growth factors, such as epidermal growth factor, are potential candidates. 
We demonstrated that epidermal growth factor stimulated ACTH secretion in primates at doses or concentrations 
equimolar to those of CRH. We also examined the in vivo effects of TNF-a and platelet activating factor (PAF). 
Both substances produced a CRH-mediated response of the HPA axis of intact rats. We also examined 
recombinant interleukin-6 (IL-6) in humans. It appears to be the most potent ACTH secretagog known. In a 
dose- and time-dependent fashion, IL-6 stimulated CRH, and additionally, parvocellular AVP, magnocellular AVP, 
pituitary ACTH and adrenal Cortisol secretion (see project ZOl HD 00615-15 DEB). 

In the search of tissue CRF(s) we have examined two models of stress. First, patients undergoing neck 
exploration or major abdominal surgery and receiving identical perioperative sedation and anesthesia were 
examined before, during, and after surgery. In the former group we saw only very low plasma concentrations 
of CRH and mild activation of the HPA axis and the adrenomedullary system during surgery. ACTH and Cortisol 
secretion was continuous rather than pulsatile and the ratio of ACTH to Cortisol decreased when compared to 
basal secretion. These results suggest both the possible presence of a long-acting tissue CRF and of an adrenal 
component in surgical stress. The major elevation of Cortisol, ACTH and epinephrine took place during 
anesthesia reversal. In contrast, in patients undergoing major surgery there was major activation of the HPA axis 
both during surgery and during recovery. In these patients, we found elevated concentrations of circulating CRH 
and profound elevations of AVP, ACTH, Cortisol, catecholamines and neuropeptide Y, a peptide which is co- 
secreted with catecholamines and which has ACTH-like properties. Interestingly, secretion of CRH and ACTH 
was pulsatile with relative concordance of the pulses which took place approximately every 30-35 min. 

Second, we examined the stress of dynamic exercise using a treadmill. Dynamic exercise is a primal component 
of the "fight or flight" response. The HPA axis as well as vasopressin, growth hormone, aldosterone, and 
prolactin secretion, are activated during exercise in an exercise intensity-dependent fashion correlating well with 
both percent of maximal 02 consumption (V02max) and plasma lactate elevations. Trained subjects could 
perform high workloads with minimal activation of the axis, a result of chronic adaptation. There was a clearcut 
relationship between the exercising muscle and the HPA axis. 

Upon CRH stimulation highly trained subjects (obligate athletes) had obvious evidence of hypercortisolism 
(blunted ACTH and Cortisol responses and high baseline Cortisol) suggesting chronic alterations of their HPA axis 
reminiscent of those seen in melancholic depression, anorexia nervosa, and chronic active alcoholism. We 
pursued this lead by psychometric testing of highly trained runners. It appears that during sustained physical 
training the affect of athletes is normal, suggesting that the self-imposed obligatory exercise in these athletes may 
be correcting some inherent mood disorder. 

To extricate potential differences in the activity of the stress system within the normal population we performed 
a 90 V02max exercise test 2h after administration of a high dose of dexamethasone (4 mg). Two populations 
of subjects were distinguished. Approximately two thirds of the subjects were completely suppressed, while one- 
third had marked ACTH, AVP, and Cortisol responses, which broke through the suppression of the glucocorticoid. 
The latter group also scored higher than the former group in the Spielberger anxiety scale. All volunteers had 
a normal single dose dexamethasone suppression test. The question arises as to whether the latter group is 
carrying a hereditary and/or constitutional trait of stress system hyperactivity and, hence, increase vulnerability 
to melancholic depression and anxiety disorders. Could the dexamethasone - exercise test be superior to 
dexamethasone or oCRH tests in revealing stress system hyperactivity? Could modifications of this combined 
test also help revealing a hypoactivity group, whose members would be vulnerable to develop atypical depression, 
chronic fatigue/fibromyalgia syndromes or autoimmune disorders (see project ZOl HD 00615-15 DEB)? 

We have developed a hypothalamic organ culture system in which we study the in vitro regulation of 
hypothalamic CRH secretion directly. We have shown that serotonin, acetylcholine and the catecholamines (alpha 
receptors) stimulate CRH secretion whereas the GABA/benzodiazepine system is inhibitory. Glucocorticoids, 



33 



ZOl HD 00618-14 DEB 

ACTH and p-endorphin, and CRH exert negative feedback (long, and short, and ultra short feedback loops, 
respectively). Alpha-MSH, and CLIP have suppressing effects on the CRH neuron. Interestingly, epidermal 
growth factor, interleukin 1, tumor necrosis factor-a, interleukin 2, interferon-y, platelet activating factor (PAF) 
and several eicosanoids show strong CRH-releasing properties. The responses to interleukin 1 and TNF-a could 
be inhibited by prostaglandin synthesis blockade. These substances may represent some of the putative links 
between the immune-inflammatory response and the endocrine system. Recently, we demonstrated that 
vasopressin is a potent stimulus of CRH secretion, an effect that could be antagonized with vasopressin receptor 
antagonists of the Vj (V,b) type. This is interesting in light of the co-presence of CRH and vasopressin in the 
paraventricular nucleus and the known synergistic effects of CRH and vasopressin on ACTH secretion. 

We have examined the effects of hypothyroidism and hyperthyroidism on the activity of the HPA axis and in 
particular the CRH neuron. We have found that they are, respectively, associated with hyposecretion and 
hypersecretion of CRH, a phenomenon that could explain the depressive symptomatology seen in both states. 
Using similar methodology, we have shown that cholecystokinin stimulates CRH secretion via peripheral vagal 
receptors and that neuropeptide Y has stimulatory effects upon the HPA axis at two levels: the hypothalamic CRH 
neuron and the adrenal cortex itself 

Pregnancy is the only known physiologic state in man in which CRH circulates in plasma at levels expected to 
cause activation of the pituitary adrenal axis (100 pg/ml up to 4000 pg/ml). These levels gradually increase even 
further during labor. Plasma CRH concentrations return to undetectable (< 15 pg/ml) following delivery. 
Interestingly, it has been known for years that the last 2-3 months of pregnancy are characterized by 
hypercortisolism at a degree similar to what has been observed in severe depression and anorexia nervosa. We 
have developed an in vitro perfusion system in which-full thickness placenta fragments are kept in culture. These 
fi-agments contain a 1300 nucleotide long mRNA which hybridizes with a CRH-specific cDNA probe. 
Glucocorticoids, prostaglandins, catecholamines, oxytocin and vasopressin have no inhibitory or stimulatory 
effects on placental secretion of CRH in this model. The latter is chromatographically identical to hypothalamic 
CRH. Our perfused placenta fragments also secrete immunoreactive beta-endorphin, a-MSH, and A-dynorphin, 
chromatographically identical to synthetic human beta-endorphin, a-MSH, and A-dynorphin, respectively. Both 
CRH and oxytocin stimulate markedly the secretion of beta-endorphin, a-MSH and A-dynorphin by the placenta. 
These findings have raised the following questions: 1) Does the high intra-pregnancy CRH suppress hypothalamic 
secretion of CRH (via Cortisol negative feedback) and therefore lead to a brief postpartum adrenal insufficiency 
state, followed by a later rebound of endogenous hypothalamic CRH secretion 6-8 weeks later? Interestingly, 
the very common "postpartum blues", occur in the first week postpartum whereas postpartum depression occurs 
1-3 months later. 2) What regulates the secretion of placental CRH? Mechanical contraction or ischemia of the 
placental may be responsible for the elevations of plasma CRH seen during labor. 3) What is the function of 
pregnancy plasma CRH other than stimulating the maternal pituitary-adrenal axis? Do the vasodilatory properties 
of CRH play a role in labor? For instance, CRH-induced superior mesenteric vessel dilation may protect the 
maternal intestinal tract from ischemia. Moreover, circulating CRH might regulate uterine blood vessel flow 
towards the placenta. The latter was recently shown convincingly by an Australian group. 

We recently completed a study examining the circadian and pulsatile activity of the HPA axis in normal pregnant 
women in the third trimester of pregnancy (32 wks, before the concentrations of CRH-BP in plasma decline). 
We found the placental CRH is secreted in a pulsatile but noncircadian fashion. Crosscorrelation demonstrated 
a positive correlation between placental CRH and ACTH and Cortisol, however the concurrent presence of the 
circadian rhythm of ACTH and Cortisol suggests that the circadian rhythmicity of ACTH and Cortisol are 
maintained by the other major ACTH secretagog, parvocellular AVP, which is also secreted in a pulsatile and 
circadian fashion, as parvocellular CRH. Naturally, levels of AVP in the systemic circulation were extremely low, 
as a result of peripheral proteolysis characteristic of the pregnant state. 

We recently completed the analysis of a prospective, longitudinal study of pregnant-postpartum women which 
included psychometric testing and CRH testing in the postpartum. Overall, all women were euthymic while 
pregnant and had a transiently suppressed CRH neuron in the postpartum. When the group that developed 



'"1 .; 



ZOl HD 00618-14 DEB 

blues/depression was compared to the women that remained euthymic, there was a clear difference between the 
two groups with the former's suppression being more profound and longstanding than the latter's. These findings 
suggest that the postpartum blues/depression syndromes should be included in the stress system hypofiinction 
states. If women during the postpartum had a defective HPA axis response to inflammatory stimuli, such as IL-6, 
this would provide an explanation for their increased vulnerability to autoimmune/inflammatory disease during 
this period of their life (see project ZOl HD 00615-15 DEB). 

C. Roles and Actions and Pathophysiology of HPA and HPG Axes Hormones 

Glucocorticoids - Glucocorticoid Resistance Syndromes: 

Glucocorticoids have multiple actions which are essential in maintenance of homeostasis in both the resting 
(unstressed) state and during stress. The effects of glucocorticoids that are important on maintenance of resting 
homeostasis have been called "permissive or normalizing". 

Glucocorticoids act upon their target tissues by modulating gene expression. This is attained by the interaction 
of a glucocorticoid-glucocorticoid receptor complex with specific sites in the nucleus, that regulate the expression 
of genes coding for function-specific proteins. We had the opportunity to study several large kindreds and 
individuals with generalized glucocorticoid resistance that had alterations of their glucocorticoid receptors, several 
New World primate species with generalized glucocorticoid and other steroid hormone resistance, and recently. 
New World prairie roles with generalized glucocorticoid resistance. We have obtained permanent cell lines from 
such individuals and species and are studying them in an attempt to understand the molecular mechanisms of 
hormone resistance and using them as an experiment of Nature to understand fundamental principles of 
glucocorticoid action. New World primates in addition to glucocorticoid resistance have progesterone, estrogen, 
aldosterone, androgen and vitamin D resistance. We demonstrated abnormalities in their glucocorticoid, progestin, 
estrogen and mineralocorticoid receptor. The animal model is of particular interest because its generalized steroid 
hormone resistance suggests a common link among the six classes of steroid hormones as they interact with their 
receptor and "acceptor" sites to modulate gene expression. 

We have cloned, sequenced and performed in vitro mutagenesis studies with the glucocorticoid receptor of the 
patients and nonhuman primates. We found two different single amino acid substitutions in the steroid binding 
domain of the glucocorticoid receptor, which were responsible for the decreased affinity and effect of the 
glucocorticoid receptor in two different propositi, that of the initial kindred and a subsequent interesting sporadic 
case. Homozygosity for the defect in the first kindred led to severe disease, whereas heterozygosity was 
associated with mild hypercortisolism, without overt clinical manifestations. The defect in our sporadic case with 
glucocorticoid resistance was a novel, de novo nonconservative amino acid substitution in the hinge region the 
glucocorticoid receptor, which not only abolished all agonist activity of the mutant glucocorticoid receptor, but 
also caused it to be a potent negative inhibitor of the normal receptor produced by the unaffected allele. The 
patient had moderate glucocorticoid resistance with hypertension and infertility. In his mid 30's, this patient 
developed also pituitary Cushing's disease. His corticotropinoma expressed P53 activity, suggesting that 
decreased glucocorticoid feedback due to glucocorticoid resistance of the corticotroph and of the hypothalamic 
neurons producing its secretagogs may be an early step in pituitary tumorigenesis. We have several additional 
families with glucocorticoid resistance whose glucocorticoid receptor we are evaluating now in great detail. Thus, 
a splice site micro deletion was found, resulting in expression of only one of the alleles, and associated with 50 
percent reduction in receptor number and hyperandrogenism in the proposita of this kindred and biochemical 
hypercortisolism in the affected brothers and father. Our results indicate that glucocorticoid resistance results 
from multiple defects and can be autosomal recessive, with heterozygotes expressing a subclinical phenotype, or 
autosomal dominant, with heterozygotes presenting with disease. We also have 2 families in whom we found 
no structural abnormality of the glucocorticoid receptor gene. We consider alternative mechanisms. 

We obtained additional evidence that glucocorticoid resistance could lead to corticotropinoma formation. One 
of 4 patients with Nelson's syndrome was found to have a somatic nonsense mutation resulting in a frame-shift 



o «_■■ 



ZOl HD 00618-14 DEB 

and a truncated, inactive glucocorticoid receptor. Interestingly, none of the 4 tumors expressed abnormal P53 
activity. 

Glucocorticoids- An Endosenous Determinant of Glucocorticoid Sensitivity: 

The human glucocorticoid receptor gene, located in the long arm of chromosome 5 consists of 10 exons. Exons 
9a and 96 can be alternatively spliced into the mRNA to encode for the classic glucocorticoid receptor a and a 
nonligand-binding receptor which differs only in the C terminus. We demonstrated that mRNA of the 6 isoform 
is expressed in all human tissues examined and that in a co-transfection system in vitro it is able to antagonize 
the effects of glucocorticoids mediated by the a isoform. We concluded that the ratio of GRa to GR6 may 
determine the sensitivity of a tissue to glucocorticoids. 

Glucocorticoids - Effects on Growth: 

We examined the influence of hypercortisolism on basal and stimulated growth hormone (GH) secretion. Young 
patients with Cushing's syndrome have profoundly suppressed spontaneous and provoked GH secretion. This 
deficiency takes over a year to correct, suggesting a rather protracted direct or indirect effect of the 
hypercortisolism on the somatotroph. Interestingly, during the convalescence year, levels of insulin-like growth 
factor I (IGF-I) in plasma remain normal, while those of IGF-binding protein 3 decrease, providing an explanation 
for the continuing growth of pediatric patients after cure of hypercortisolism, even though the levels of GH are 
suppressed. We found that patients who had Cushing's syndrome during their growth years lost on the average 
7 cm of their final height, when they were followed up as adults. 

Glucocorticoids - HPA Axis Feedback Effects via Type I Receptors: 

The glucocorticoid receptor type I (mineralocorticoid receptor) is profoundly involved in the feedback regulation 
of the HPA axis in the rat. We demonstrated that the mRNA of this peptide is also expressed in Old and New 
World primates, in similar regions of the CNS. We examined its importance by administering specific type I 
antagonists to normal humans but found no effect of such antagonists on basal and CRH-stimulated Cortisol 
secretion. 

Sleep and b-Sleep-Inducins Peptide in Cushing's Syndrome: 

Patients with Gushing syndrome suffer from sleep disorders. We have developed an assay for 5-sleep-inducing 
peptide to study these patients in synchrony with continuous EEG monitoring. Preliminary study demonstrates 
that although 5-sleep induced peptide immunoreactivity is decreased in Gushing syndrome, its relationship with 
the sleep disturbances observed in this condition is tenuous. 

Aldosterone Resistance-Pseudohypoaldosteronism: 

Pseudohypoaldosteronism is a congenital condition characterized by profound salt loss, hypokalemia and acidosis 
in the presence of extremely elevated levels of plasma and urinary aldosterone. The resistance to aldosterone can 
be limited to the kidney or generalized to all mineralocorticoid target tissues. The clinical severity of the 
syndrome improves with age. About 2/3 of the cases are sporadic, while 1/2 are familial. We studied several 
sporadic cases for the presence of defects in the mineralocorticoid receptor to find no pathogenetic mutations. 
We have focused our efforts on a postreceptor mechanism, hopefully to find mutations in one of the 4 subunits 
of the amiloride-sensitive sodium channel. 



n r 



ZOl HD 00618-14 DEB 

ACTH Resistance - Hereditary Isolated Glucocorticoid Deficiency. 

ACTH regulates glucocorticoid secretion by the adrenal cortex via its recently cloned receptor. This membrane 
protein belongs to the superfamily of G protein-coupled receptors, forming its own distinct class with the 
receptors for aMSH. We mapped the gene of the ACTH receptor in the short arm of chromosome 1 8 and studied 
families with hereditary isolated glucocorticoid deficiency (congenital insensitivity to ACTH) for potential defects 
of this gene. We found that point mutations resulting in premature termination of the receptor or nonconservative 
amino acid substitutions in key functional areas of the molecule cause the disease. Heterozygote carriers are 
clinically healthy but revealed by CRH testing, since they have markedly exaggerated responses of ACTH to this 
hormone. 

Testicular LH Resistance - Leydis Cell Hypoplasia - Ovarian LH Resistance: 

LH regulates testosterone secretion by Leydig cells and ovulation and corpus luteum formation by the ovarian 
follicle via its recently cloned receptor. This membrane protein belongs to the superfamily of G protein coupled 
receptors, forming its own distinct class with the receptors for the other glycoprotein hormones. This group 
studied families with hereditary primary Leydig cell failure with genetic male members that presented with 
phenotypically female external genitalia or micropenis and genetic females with primary amenorrhea and 
infertility. They found that point mutations resulting in premature termination of the receptor or nonconservative 
amino acid substitutions in key functional areas of the receptor molecule caused the disease. Heterozygotes were 
clinically healthy. Genetic females with complete absence of the LH receptor developed normally in utero and 
had a normal pubertal development, but remained anovulatory. 

D. Mechanisms of Adrenocortical Tumorigenesis 

We have studied plausible mechanisms of adrenocortical tumorigenesis with interesting preliminary results. All 
tumors are monoclonal. About one third of malignant adrenocortical tumors and both available adrenocortical 
cancer cell lines have abnormalities of the p53 gene, while none, including the two cell lines and a series of 
adenomas has activating mutations of the ACTH receptor gene or mutations of the G proteins. 

In our patient population, we had 5 kindreds and several sporadic cases with primary pigmented nodular 
adrenocortical disease (PPNAD) or micronodular adrenal disease, a major component of Carney complex, a 
multiple neoplasia/lentiginosis syndrome transmitted in an autosomal dominant fashion. In collaboration with J.A. 
Carney of the Mayo Clinc, we included an additional 6 kindreds in our series and attempted chromosomal 
mapping of the syndrome by linkage analysis. We indeed mapped the defective gene of the complex within a 
~6 cM region of the short arm of chromosome 2 in band pl6. A small number of genes are known in this area, 
such as those of calcineurin B and two DNA repair enzymes, previously shown to be involved in nonpolyposis 
colon cancer and/or microsatellite instability. 

Significance to Biomedical Research and the Program of the Institute : 

A. Clinical Applications and Implications of CRH 

Different patterns of plasma ACTH and Cortisol responses to CRH have been noted in different diseases, including 
hypercortisolism and hypocortisolism. The CRH stimulation test appears to differentiate between Cushing's 
disease and the ectopic ACTH syndrome. In addition, it appears to be of diagnostic value in distinguishing 
between Cushing's disease and hypercortisolism due to depression. Because of its biological properties hCRH 
is an important means to study the normal physiology of the hypothalamic-pituitary-adrenal axis. In psychiatric 
diseases characterized by hypercortisolism, such as melancholic depression and anorexia nervosa, a 
pathophysiologic role has been suggested for CRH and this hypothesis is currently tested. Were it true, new 
treatments could be developed with major goal to prevent CRH hypersecretion or action. We plan to examine 






I 



ZOl HD 00618-14 DEB 

the efficacy of a newly discovered nonpeptide CRH antagonist, which could be used orally and which is expected 
to cross the blood brain barrier. 

Our recent studies with atypical depression, the chronic fatigue/fibromyalgia syndromes and the postpartum 
blues/depression syndromes suggest that a spectrum of human pathologic conditions exist in which the central 
CRH system is hypofunctional. Activation of the CRH system in such patients may help with the resolution of 
the clinical picture. Also, CRH agonists or molecules that displace CRH from its binding protein might be useful 
in the treatment of these conditions. We study the psychological, social and cognitive changes in patients with 
Cushing's syndrome before and after cure of their hypercortisolism. It is of interest that we have found 
similarities between Cushing's syndrome both before and after cure and atypical depression, probably stemming 
from the same pathologic decrease in CRH secretion. We found that the common marmoset could be an excellent 
model of human depression and psychosocial retardation. We found that sexual abuse in childhood has profound 
prolonged effects on the hypothalamic-pituitary-adrenal axis, as well as on the affect of these individuals. 

B. Regulation of the Hypothalamic-Pituitary-Adrenal Axis In Vivo and In Vitro 

We determined the changes of the HPA axis during the stress of surgery or exercise. We have also demonstrated 
that hyperthyroidism and hypothyroidism are respectively, associated with hypersecretion and hyposecretion of 
CRH which might explain the presence of depressive symptomatology in both conditions. Several peripheral 
substances secreted during stress (interleukin 1 and 6, TNF-a, epidermal growth factor, platelet activating factor, 
prostanoids) appear to be CRH secretagogs of physiologic relevance. We determined the major stimulatory 
(serotonin, acetylcholine, norepinephrine) and inhibitory (GABA/benzodiazepine system) neurotransmitters of 
hypothalamic CRH secretion in vitro and demonstrated the presence of a long, a short and an ultra-short feedback 
loop respectively with glucocorticoids, ACTH and p-endorphin, and CRH, all playing a role at the level of the 
CRH neuron. These advances from basic science are helpful in understanding the various disorders of the HPA 
axis that we study. We have demonstrated that the human placenta secretes large quantities of CRH, POMC- 
derived peptides (primarily 6 endorphin and a-MSH) and dynorphin A and that CRH exerts autocrine and/or 
paracrine actions on the placenta by causing 6-endorphin, ot-MSH and dynorphin A secretion by this tissue. 
These results may influence our way of thinking about the physiology and pathophysiology of pregnancy and 
labor and delivery. We confirmed our hypothesis that the postpartum blues/depression represent a state of 
"adrenal suppression" characterized by suppression of hypothalamic CRH secretion and symptoms of atypical 
depression. We have examined in great detail the regulatory region of the human CRH gene. 

C. Roles, Actions, and Pathophysiology of HPA and HPG Axis Hormones 

Pathologic mutations and/or deletions of the glucocorticoid receptor gene are responsible for human familial 
glucocorticoid resistance. Generalized or tissue-specific resistance can lead to human tumorigenesis. The ratio 
of the two isoforms of the glucocorticoid receptor may define a tissue's sensitivity or resistance to 
glucocorticoidds. The glucocorticoid receptor isoforms may participate in the translocation of AIDS virus 
proteins, such as vpr, which by activating the glucocorticoid receptor may stimulate viral replication via the GREs 
present in the promoter region of HIV. Also, by exerting glucocorticoid effects, the glucocorticoid receptor - vpr 
complex may cause further immunosuppression. Thus, the glucocorticoid receptor may participate in the hallmark 
manifestations of AIDS, may hasten the conversion from the carrier to the disease state and may curtail the 
terminal phase of the disease. 

A transcription or other factor common to all steroid hormones may be defective in New World primates. We 
suggest that the glucocorticoid receptor may be involved in highly prevalent human diseases, such as obesity, 
hypertension, depressive/anxiety syndromes and autoimmune diseases by providing excessive or defective 
glucocorticoid activity to a tissue. 

It is important to pursue our search for the molecular defect of pseudohypoaldosteronism. We have defined 
common human mineralocorticoid receptor and amiloride-sensitive sodium channel subunit a polymorphisms 



36 



ZOl HD 00618-14 DEB 

which may be useful in the pursuit of the genetic determinants of hypertension. 

We have examined several families with hereditary isolated glucocorticoid deficiency (ACTH resistance) for 
possible defects of the ACTH receptor gene. We found pathologic mutations, altering the receptor and causing 
the disease. The heterozygote parents and grandparents have abnormal ACTH responses to ovine corticotropin 
releasing hormone, suggesting that this test can be employed for the ascertainment of the carrier state in this 
disorder. We found that the triple A syndrome, which has hereditary isolated glucocorticoid deficiency as a 
component (Adrenal insufficiency, Achalasia, Alacrima), is not associated with molecular defects of the ACTH 
receptor. 

We have defined the LH receptor gene as a locus of the defect in complete male pseudohermaphroditism (Leydig 
cell hypoplasia) and micropenis in genetic males and in anovulation and primary amenorrhea in genetic females. 

D. Mechanisms of Adrenocortical Tumorigenesis 

Adrenal cancer continues to represent a rare but aggressive carcinoma that affects children and young individuals 
and responds little in terms of medical therapy. Every effort should be placed in understanding this disease, in 
achieving early diagnosis and in devising effective cures. 

Our Carney complex research may not only elucidate the molecular pathophysiology of a rare multiple 
neoplasia/lentiginosis syndrome that afflicts children and young individuals but also may help unravel important 
physiological processes and mechanisms of tumorigenesis, as it has happened already with other rare syndromes. 

Proposed Course: 

A. Clinical Applications and Implications of CRH 

1. Is CRH Involved in the Pathophysiolosy of Affective Disorder? 

a) We have formulated the general hypothesis that CRH plays a pathophysiologic role in psychiatric disease 
characterized by hypersecretion or hyposecretion of this peptide. CRH antagonists or inhibitors of CRH secretion 
as well as CRH agonists or stimulants of CRH secretion, will be used as tools to test this hypothesis. Animal 
models for depression would be ideal to begin such testing. 

b) It is evident from studies by S. Levine and S. Suomi that neonatal separation of rats or monkeys from their 
mothers can be associated with hyperactivity of the hypothalamic-pituitary-adrenal axis and behavioral 
disturbances throughout their life. We obtained similar results in sexually abused girls long after the abuse took 
place. These findings suggest that there are critical periods in human life as well, associated with permanent 
alterations in the reactivity to stress. The hypothesis that CRH is involved in this early sensitization can be 
examined in various ways. We plan to use CRH antagonists to reverse the clinical picture of animals that develop 
the syndrome. 

2. Can We Unravel Affective Disorder Traits? 

If for genetic or constitutional reasons the number of hypothalamic CRH neurons in patients prone to develop 
melancholic depression/anxiety disorders were increased, one would hope that a strong stimulus of the CRH 
neuron, such as IL-6, would be able to distinguish this group from normal controls. The same may be true for 
our dexamethasone-exercise test paradigm. Similarily, if for the same reasons the number of hypothalamic CRH 
neurons in patients prone to develop atypical depression, the chronic fatigue/fibromyalgia syndrome or 
autoimmune disease, were decreased, lL-6 might also be able to detect them. 






o 



ZOl HD 00618-14 DEB 

3. Differential Diagnosis of Cushins's Disease from Pseudo-Ciishins States 

In spite of the assistance of the combined dexamethasone suppression/oCRH stimulation test, it is still difficult 
to differentiate some patients with depression and hypercortisolism from patients with mild Cushing's disease. 
Based on previously obtained information from human and in vivo and in vitro studies, we have designed a study 
which may assist us further with this differential. 

Thus, the cytokine IL-6 administered acutely should stimulate the HPA axis of patients with pseudoCushing's 
(depression, chronic active alcoholism) but not that of patients with Cushing's syndrome of any etiology since 
in all instances hypothalamic CRH should be suppressed. 

4. Postpartum "Blues" and Postpartum Depression 

Following delivery of the fetus and the placenta, the mother loses a source of peripheral CRH production. This 
is likely to be associated with subphysiological Cortisol secretion during the first days of the postpartum period, 
a result of a suppressed hypothalamic CRH neuron. Interestingly, 40-50% of postpartum women develop the 
postpartum blues during the first 2-3 weeks postpartum. We hypothesized that this syndrome was equivalent to 
glucocorticoid withdrawal, in a sense being an analogue of the steroid withdrawal syndrome of the post 
transsphenoidal surgery patient with Cushing's disease, who despite glucocorticoid replacement develops 
"postoperative blues". 

Three weeks to three months postpartum, approximately 10% of women develop a frank form of depression which 
interferes with their overall function. Interestingly, this is the period of the putative hypothalamic CRH recovery. 
Our prospective study showed the alterations of the hypothalamic-pituitary-adrenal axis in the postpartum and the 
suppression observed was related to mood changes in the mothers. We anticipate that high levels of plasma CRH 
or high urinary free Cortisol excretion pre-partum might predict the severity of the postpartum blues or depression 
syndrome. A larger study needs to be performed now to test this hypothesis. In a small subgroup, lL-6 should 
be administered, as discussed in project ZOl HD 00615-15 DEB, to test the hypothesis of diminished 
responsiveness to inflammatory stimuli in this period, which is characterized by increased vulnerability to 
autoimmune disease. 

Growth and development of infants of postpartum depressed mothers might be affected to a degree proportional 
to the severity of the maternal syndrome. Interestingly, maternal circulating CRH and cord blood CRH 
concentrations are positively correlated. Thus, the infants should be followed as well. 

B. Regulation of the Hvpothalamic-Pituitary-Adrenal Axis In Vivo and In Vitro 

1. Resulation of the Circadian Rhythmicity and Stress Activation of the Human and Rat Hvpothalamic-Pituitary- 
Adrenal Axis 

We have shown that continuous i.v. infusion of pharmacologic amounts of oCRH in normal volunteers leads to 
mild hypercortisolism (UFC 150-200 mcg/d) and preservation of the plasma ACTH and Cortisol circadian rhythm. 
Interestingly, the secretory episodes of ACTH and Cortisol continue to be seen despite the maximal circulating 
concentration, of CRH. On the other hand, CRH concentrations in the CSF have a circadian rhythm which is in 
opposite phase to the circadian rhythm of plasma Cortisol concentrations. We have postulated that ACTH 
secretagogues other than CRH may be involved in the regulation of episodic secretion of ACTH and Cortisol and 
the circadian rhythmicity of the HPA axis. Vasopressin is a such potential factor. We have available several (V3) 
receptor vasopressin antagonists which will be administered continuously i.v. to test the hypothesis that 
vasopressin participates in the generation of the circadian rhythm. We have also generated antibodies against 
vasopressin and CRH with which we perform immunoneutralization studies in experimental animals. 



4.. 



Jtl 



n 



ZOl HD 00618-14 DEB 

The V3 receptor antagonists could be employed to define the degree of vasopressin participation in the stress- 
related activation of the pituitary-adrenal axis in man. Thus, graded levels of exercise, or an insulin tolerance 
test, could take place while a vasopressin antagonist infusion is being administered. 

2. Regulation of Placental CRH. POMC-Derived Peptides and Dynorphin A In Vivo and In Vitro 

The possible association of placental CRH secretory episodes with uterine contractions will be examined by 
frequent, every 3 min serial sampling over 3 hours in end-stage labor. 

Prostanoids and platelet activating factor will be examined as stimulants of placental CRH or POMC-derived 
peptides in vitro . Of particular interest are also several agents, including serotonin, epidermal growth factor 
(EGF), platelet derived growth factor (PDGF), interleukins 1 and 6, and tumor necrosis factor (cachexin), which 
cause hypothalamic CRH secretion in vitro . PDGF was recently found to mimic the effect of serum from 
preeclamptic women in causing lysis of cultured endothelial cells. It is of interest that preeclampsia is associated 
with elevated circulating CRH concentrations in pregnancy. 

C. Roles, Actions and Pathophvsiology of HPA and HPG Axes Hormones 

1. Clonins of the Glucocorticoid Receptor Type II Gene in Patients with Glucocorticoid Resistance and in Animal 
Models 

We are now examining the glucocorticoid receptor of members of several new families with glucocorticoid 
resistance. Also, we are pursuing several hypotheses for the mechanism(s) of glucocorticoid resistance in the 2 
families in which no structural abnormalities of the glucocorticoid receptor gene were found. The aldosterone, 
progesterone, and estrogen receptor of the New World primates will be cloned also, with the assistance of probes 
obtained from the human. We postulate that the common link between all steroid hormones that is responsible 
for the generalized steroid hormone resistance in New World primates represents a common modulator of all 
steroid receptors. This could be an enzyme, such as a steroid receptor phosphokinase, a common trans-activating 
factor necessary for the interaction of the steroid receptor in the nucleus with their responsive elements, or a 
common steroid-class specific binding protein. We are characterizing the prairie vole as a glucocorticoid resistant 
animal and examine the molecular mechanisms of resistance in this species, as well. 

2. Continuing Studies with the 6-Isoform of the Glucocorticoid Receptor 

There are many important questions remaining in this area. We need to demonstrate and quantify the actual 
protein content of each isoform in human tissues. We need to demonstrate the actual location of the 6 isoform 
within the cell and its relation to heat shock proteins. We need to examine the interaction of the 6 isoform not 
only with GREs, but also with transcription factors known to interact with glucocorticoid receptors and known 
to be influenced by this interaction. These include the cjun-cfos and the NF-kB/Rel A heterodimers. One could 
include here the HIV protein vpr, which apparently binds to the glucocorticoid receptor and translocates with it 
into the nucleus to potentially enhance viral replication and suppress host immune functions v]a classic 
glucocorticoid receptor-mediated pathways. This hypothesis is testable and we plan to pursue it. We will pursue 
also the potential involvement of GR6 in human pathophysiology. Thus, we will examine tissues of patients with 
glucocorticoid-resistant asthma and other conditions for overexpression of the 6-isoform, and tissues of patients 
with glucocorticoid hypersensitivity (metabolic syndrome X) for underexpression of the 6-isoform. 

3. Molecular Mechanisms of Aldosterone Resistance 

We are pursuing a likely postreceptor site, the amiloride-sensitive sodium channel, with its 4 known subunits. 



ZOl HD 00618-14 DEB 



4. ACTH Receptor Studies in Patients with ACTH Resistance 



We are now examining the ACTH receptor in several additional kindreds with isolated hereditary ACTH 
resistance, as well as kindreds with the triple A syndrome (Adrenal insufficiency, Achalasia, Alacrima). In the 
latter we will first attempt genetic linkage studies, once we have collected the necessary number of subjects. 

5. LH Receptor Studies in Patients with LH Resistance 

We are examining other potential candidates with defects of the LH receptor. 

D. Mechanisms of Adrenocortical Tumorigenesis 

We continue to examine major candidate genes potentially participating in adrenocortical tumorigenesis. These 
include those of pi 6, steroidogenetic factor 1 and DAX 1. 

We continue our two-prong approach towards elucidating the molecular pathophysiology of Carney complex. 
Thus, we examine candidate genes within the 2pl6 region, in which we localized the gene and continue to cone 
down on a progressively smaller region, until we hopefully identify the actual gene. 

Protocols: 

Human 



82-CH-45 



Chrousos Dose-Response Relationship for Single Doses of Corticotropin- 

Releasing Hormone (CRH) in Normal Volunteers and in Patients with 
Adrenal Insufficiency (active) 

This blanket protocol enables us to administer ovine corticotropin- 
releasing hormone (CRH) as a provocative test of the hypothalamic- 
pituitary-adrenal (HPA) axis in men, women, and children with 
potential alterations of this axis. The peptide continues to be an 
investigational drug (IND# 19802) and all data concerning its 
administration are captured and reported annually to the FDA. During 
the past year oCRH was administered under this and other approved 
protocols. 



82-CH-125 



86-CH-172 



Gold 



Laue 



Studies of Corticotropin Releasing Factors (CRF) and Related 
Neurotransmitters, Neurotransmitter Metabolites and Peptides in the 
Spinal Fluid of Patients with Cushing's Disease (completed) 

Dose Response Relationships between Exogenous ACTH and Adrenal 
Cortisol Secretion in Normal Volunteers and Patients with 
Abnormalities of the Hypothalamic-Pituitary-Adrenal Axis 
(completed) 



88-CH-120 



Chrousos The Hypothalamic-Pituitary-Adrenal Axis in Pregnancy, the 

Postpartum Period, and in Postpartum Depression Syndromes 
(completed) 



m 



88-CH-115 



90-CH-125 



90-CH-124 



90-CH-194 



91-CH-166 



92-CH-181 



94-CH-134 



ZOl HD 00618-14 DEB 

Chrousos Placental Secretion of Corticotropin Releasing Hormone during 

Pregnancy and Labor and Delivery (partially completed) 

Chrousos Psychological, Social and Cognitive Changes in Children, Adolescents 

and Adults with Cushing syndrome (completed) 

Friedman Circulating Hormones and EEG Monitoring in Patients with Cushing's 

Syndrome (completed) 

Yanovski Inferior Petrosal Sinus Sampling for the Determination of 

ACTH Concentration in Normal Volunteers and in Patients with 
Disorders of the Hypothalamic-Pituitary-Adrenal Axis (active) 

This protocol allows a procedure which is diagnostic in Cushing's 
syndrome to be performed in normal volunteers and patients with 
abnormalities of the HPA axis, whom it would not benefit. This 
protocol, thus, provides valuable normal control samples and samples 
that may help us understand better the pathophysiology of the HPA 
axis. 

Chrousos Hypothalamic-Pituitary-Adrenal Axis Function in Offspring of 

Affectively III Mothers vs Offspring of Normal Control Mothers 
(completed) 

Peeke Energy Metabolism in Patients with Seasonal Affective Disorder 

(completed) 

Papanicolaou Dose-Response Relationships for Single Doses of Recombinant 
Interleukin-6 in Normal Volunteers and Patients with Disorders of the 
Hypothalamic-Pituitary-Adrenal Axis (active) 

This protocol concerns the administration of recombinant interleukin-6 
to normal volunteers and patients with disorders of the HPA axis. 
Interleukin-6 is an investigational drug (IND# 5419), with which we 
have already obtained some experience from a phase 1 study of 
cancer patients done by NCI. A dose-response curve in normal 
volunteers has been completed and a dose has been selected to be 
administered to the various patient groups listed in the protocol. The 
hypothesis of the protocol is that as a hypothalamic stimulus of CRH, 
acute administration of interleukin-6 will distinguish biochemically 
not only patients with a clearly disordered HPA axis, i.e. Cushing's 
vs. pseudoCushings's vs^ melancholic depression ys^ chronic active 
alcoholism or melancholic ys^ atypical depression, but also premorbid 
states of vulnerability, i.e. individuals prone to melancholic depression 
or anxiety disorders or individuals prone to atypical depression, 
chronic fatigue/fibromyalgia, postpartum blues or depression, 
alcoholism or autoimmune disorders. 



95-CH-59 



Stratakis Definition of the Genotype and Clinical Phenotype of Primary 

Pigmented Adrenocortical Disease and Its Associated Conditions 
(Carney Complex) 



u 



ZOl HD 00618-14 DEB 

This protocol allows the examination and obtaining of samples from 
patients with Carney Complex and their families. Samples include 
blood for leukocytes and DNA and tumors for cytogenetic and DNA 
studies. The first phase of the protocol, to collect sufficient numbers 
of samples to perform the initial mapping of the defective gene by 
linkage analysis has been completed. More precise localization and 
testing of candidate genes and examination of tumors for clues 
continue. 



Animal 



-Oil rat 



89-036 rat 



Chrousos Effect of Thyroid Hormones on Hypothalamic-Pituitary-Adrenal 

Function (completed) 

Chrousos Regulation of Hypothalamic CRH & Pituitary ACTH Release (active) 



91-025 rat 



92-031 rat 



93-037 rat 



Chrousos Heat Shock Protein 90 (HSP 90) and Glucocorticoid Receptor Gene 

Expression In Vivo: Effects of Adrenalectomy and Glucocorticoid 
Administration (completed) 

Chrousos The Effects of Acute and Chronic Stress on Hypothalamic-Pituitary- 

Adrenal Axis and Sympathetic Nervous System Function of the 
Zucker (fa/fa) Rat (completed) 

Cizza Regulation of the Hypothalamic-Pituitary-Adrenal Axis at Basal 

Conditions and During Immobilization in the Aged Male and Female 
Fischer 344/N Rat (active) 



Publications: 

Aral K, Chrousos GP. Glucocorticoid resistance, Baillere's Clin Endocrinol Metab 1994;8:317-31. 

Aral K, Chrousos GP. Syndromes of glucocorticoid and mineralocorticoid resistance. Steroids 1994;60:173-9. 

Aral K, Tsigos C, Suzuki Y, Irony I, Karl M, Listwak S, Chrousos GP. Physiological and molecular aspects of 
mineralocorticoid receptor action in pseudohypoaldosteronism: a responsiveness test and therapy, J Clin 
Endocrinol Metab 1994;79:1019-23. 

Aral K, Tsigos C, Suzuki Y, Listwak S, Zachman K, Zangeneh F, Rapaport R, Chanoine JP, Chrousos GP. No 
apparent mineralocorticoid receptor defect in a series of sporadic cases of pseudohypoaldosteronism, J Clin 
Endocrinol Metab 1995;80:814-7. 

Auchus RJ, Mastorakos G, Friedman TC, Chrousos GP. Corticotropin-releasing hormone production by a small 
cell carcinoma in a patient with ACTH-dependent Gushing syndrome, J Endocrinol Invest 1994;17:447-52. 

Bamberger CM, Bamberger AM, De Castro M, Chrousos GP. Glucocorticoid receptor-beta, a potential 
endogenous inhibitor of glucocorticoid action in humans, J Clin Invest 1995;95:2435-41. 

Bamberger CM, Chrousos G.P. Glucocorticoid resistance. [Letter to the Editor]. Ann Inter Med 1994;102:1047- 
8. 



d... 



ZOl HD 00618-14 DEB 

Beuschlein F, Reincke M, Karl M, Travis WD, Jaursch-Hancke C, Abdelhamid S, Chrousos GP, Allolio B. Clonal 
composition of human adrenocortical neoplasms, Cancer Res 1994;54:4927-32. 

Bemadini R, Ciiiarenza A, Kamilaris TC, Renaud N, Lempereur L, Demitrack M, Gold PW, Chrousos GP. In 
vivo and in vitro effects of arginine-vasopressin receptor antagonists on the hypothalamic-pituitary-adrenal axis 
in the rat, Neuroendocrinology 1994;60:503-8. 

Chen H, Doppman JL, Chrousos GP, Norton JA, Nieman LK, Udelsman R. ACTH-secreting 
pheochromocytomas: the exception to the rule, Surgery, in press. 

Chrousos GP. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation, N Engl J Med 
1995;332:1351-62. 

Chrousos GP. Organization and integration of the endocrine system. In: Sperling M, ed. Pediatric 
endocrinology. Philadelphia: Saunders Co, in press. 

Cizza G, Gold PW, Chrousos GP. The stress-induced inhibition of the hypothalamic-pituitary-thyroid axis is 
attenuated in the aged Fischer 344/N male rat, Neuroendocrinology in press. 

Cizza G, Kalogeras AE, Brady LS, Bagdy G, Bergamini E, Blackman MR, Chrousos GP, Gold PW. Male 344 
Fischer rats show a progressive central impairment of the hypothalamic-pituitary-adrenal-axis with advancing age, 
Endocrinology 1994;134:1611-20. 

Cizza G, Pacak K, Kvetnansky R, Palkovits M, Goldstein DS, Brady LS, Fukuhara K, Bergamini E, Kopin IJ, 
Blackman MR, Chrousos GP, Gold PW. Decreased stress responsivity of the central and peripheral 
catecholaminergic systems in the aged 344/N Fischer rat, J Clin Invest 1995;95:1217-24. 

Crofford LJ, Pillemer SR, Kalogeras KT, Cash JM, Michelson D, Kling MA, Sternberg EM, Gold PW, Chrousos 
GP, Wilder RL. Hypothalamic-pituitary-adrenal-axis perturbation in patients with fibromyalgia, Arthr Rheum 
1994;37:1583-92. 

DeBellis M, Chrousos GP, Dom L, Burke L, Helmers K, Kling MA, Trickett PK, Putnam FW. Hypothalamic- 
pituitary-adrenal axis dysregulation in sexually abused girls, J Clin Endocrinol Metab 1994;78:249-55. 

Doppman JL, Nieman LK, Chang R, Yanovski JA, Cutler GB Jr, Chrousos GP, Oldfield EH. Selective venous 
sampling from the cavernous sinuses is not a more reliable technique than sampling from the inferior petrosal 
sinuses in Gushing syndrome, J Clin Endocrinol Metab 1995;80:2485-9. 

Doppman J, Nieman LK, Cutler GB Jr, Chrousos GP, Fraker DL, Norton JA, Jensen RT. Adrenocorticotropin 
hormone-secreting islet cell tumors: are they always malignant? Radiology 1994;190:59-64. 

Dom LD, Burgess ES, Dichek HL, Putnam F, Chrousos GP, Gold PW. Thyroid hormone concentrations in 
depressed and non-depressed adolescents: group differences and behavioral relations, J Am Acad Child Adolesc 
Psychiatry, in press. 

Dom LD, Burgess ES, Dubbert B, Kling M, Gold PW, Chrousos GP. Psychopathology in patients with 
endogenous Gushing syndrome: "atypical" or "melancholic" features, Clin Endocrinol, in press. 

Flack MR, Chrousos GP. Neoplasms of the adrenal cortex. In: Holland R, et al., eds. Cancer medicine. 4th 
ed. New York: Lea and Fibinger, in press. 






ZOl HD 00618-14 DEB 

Friedman TC, Chrousos GP. Transsphenoidal resection in Cushing's disease: definition of success. [Letter to 
the Editor]. Clin Endocrinol 1995;39:701. 

Friedman TC, Garcia-Borreguero D, Hardwick D, Akuete C, Doppman J, Dom L, Barker C, Chrousos GP. 
Decreased delta sleep and plasma delta sleep-inducing peptide immunoreactivity in patients with Gushing 
syndrome, Neuroendocrinology 1994;60:626-34. 

Friedman TC, Garcia-Borreguero D, Hardwick D, Akuete CN, Stambuk MK, Dom L, Cutler GB Jr, Nieman LK, 
Chrousos GP. Diurnal rhythm of plasma delta sleep-inducing peptide in humans: evidence for positive correlation 
with body temperature and negative correlation with REM slow wave sleep, J Clin Endocrinol Metab 
1994;78:1085-9. 

Heinrichs C, Deschepper J, Tsigos C, Drews R, Collu R, Dugardeyn C, Goyens P, Ghanem GE, Bosson D, 
Chrousos GP, Van Vliet G. Familial adrenocorticotropin unresponsiveness associated with alacrima and achalasia: 
biochemical and molecular studies in two siblings with clinical heterogeneity, European J Peds 1995;154:191-6. 

Johnson EO, Decker L, Gold PW, Chrousos GP. Distribution of hippocampal mineralocorticoid and 
glucocorticoid receptor mRNA in a glucocorticoid resistant nonhuman primate. Steroids, in press. 

Johnson EO, Kamilaris TC, Calogero AE, Gold PW, Chrousos GP. Effects of early parenting on growth and 
development in a small primate, Ped Res, in press. 

Johnson EO, Kamilaris TC, Carter CS, Calogero AE, Gold PW, Chrousos GP. The biobehavioral consequences 
of psychogenic stress in a small social primate ( Callithrix jacchus jacchus) , Biol Psych, in press. 

Karl M, Chrousos GP. Experimental and clinical models of CRH-induced pituitary tumors. In: Melmed S, ed. 
Oncogenesis and molecular biology of pituitary tumors. New York: Karger, in press. 

Karl M, Schulte HM, Chrousos GP. Mutation analysis of steroid hormone receptors. In: deKloet ER, Suntanto 
W, eds. Neurobiology of steroids. Methods in neurosciences (General Editor: Conn PM). Philadelphia: 
Academic Press, 1994;22:226-41. 

Karl M, Von Wichert G, Kempter E, Katz DA, Reincke M, Monig H, Ali lU, Stratakis CA, Oldfield EH, 
Chrousos GP, Schulte HM. Nelson's syndrome associated with a somatic frame shift mutation in the 
glucocorticoid receptor gene, J Clin Endocrinol Metab, in press. 

Latronico AC, Reincke M, Mendonca BB, Arai K, Mora P, Allolio B, Wajchenberg BL, Chrousos GP, Tsigos 
C. No evidence for oncogenic mutations in the adrenocorticotropin receptor (ACTH-R) gene in human 
adrenocortical neoplasms, J Clin Endocrinol Metab 1995;80:875-7. 

Magiakou MA, Chrousos GP. Corticosteroid therapy, nonendocrine disease, and corticosteroid withdrawal. In: 
Bardin W, ed. Current therapy in endocrinology and metabolism. St Louis: Mosby-Year Book Inc, 1994;120-4. 

Magiakou MA, Chrousos GP. Diagnosis and treatment of Cushing's disease. In: Imura H, ed. The pituitary 
gland. 2nd ed. New York: Raven Press, I994;49 1-508. 

Magiakou MA, Chrousos GP. Glucocorticoid therapy and withdrawal. In: Bone RC, ed. Current practice of 
medicine, in press. 

Magiakou MA, Mastorakos G, Chrousos GP. Final stature in patients with endogenous Gushing syndrome, J Clin 
Endocrinol Metab 1994;79:1082-5. 



4U 



ZOl HD 00618-14 DEB 

Magiakou MA, Mastorakos G, Gomez MT, Rose SR, Chrousos GP. Suppressed spontaneous and stimulated 
growth hormone secretion in patients with Gushing disease before and after surgical cure, J Clin Endocrinol Metab 
1994;78:131-7. 

Magiakou MA, Mastorakos G, Oldfield EH, Gomez MT, Doppman JL, Cutler GB Jr, Nieman LK, Chrousos GP. 
Gushing syndrome in children and adolescents: presentation, diagnosis and therapy, N Engl J Med 1994:331:629- 
36. 

Margioris A, Gravanis A, Chrousos GP. Glucocorticoids and mineralocorticoids. In: Brody T, Lamer J, 
Minneman K, Neu HC, eds. Human pharmacology: molecular to clinical. St. Louis: Mosby- Yearbook, 1994;473- 
81. 

Mastorakos G, Chrousos GP. Adrenal hyperandrogenism. In: Adashi E, Rock J, Rosenwaks Z, eds. 
Reproductive endocrinology, surgery, and technology. New York: Raven Press, in press. 

Mastorakos G, Cizza G, Kvetnansky R, Bergamini E, Blackman MR, Gold PW, Chrousos GP. Aging and acute 
stress decrease corticotropin releasing hormone in the ovary of the Fischer 344/N rat, Life Sci 1995;56:1065-71. 

Michelson D, Chrousos GP, Gold PW. Type 1 glucocorticoid receptor blockade does not affect baseline or ovine 
corticotropin-releasing hormone-stimulated adrenocorticotropic hormone and Cortisol secretion, 
NeurolmmunoModulation 1 994; 1 :274-7. 

Pacak K, McCarty R, Palkovitz M, Cizza G, Kopin IJ, Goldstein D, Chrousos GP. Decreased central and 
peripheral catecholaminergic activation in obese Zucker rats. Endocrinology, in press. 

Patchev VK, Brady LS, Karl M, Chrousos GP. Gene expression of hsp90 and glucocorticoid receptors in the 
brain: evidence for coordinate regulation by adrenal steroid levels in vivo. Cell Mol Endocrinol 1994;103:57-64. 

Patchev VK, Karalis K, Chrousos GP. Effect of excitatory amino acid transmitters on hypothalamic corticotropin- 
releasing hormone (CRH) and arginine-vasopressin release: implications for pituitary-adrenal regulation, Brain 
Res 1994;633:312-6. 

Peeke PM, Chrousos GP. Hypercortisolism and obesity, Ann NY Acad Sci, in press. 

Petrides JS, Mueller GP, Kalogeras KT, Chrousos GP, Gold PW, Deuster PA. Exercise-induced activation of the 
hypothalamic-pituitary-adrenal axis: marked differences in the sensitivity to glucocorticoid suppression, J Clin 
Endocrinol Metab 1994;79:377-83. 

Ram Z, Nieman LK, Cutler GB Jr, Chrousos GP, Doppman J, Oldfield EH. Early repeat surgery for persistent 
Cushing's disease, J Neurosurg 1994;80:37-45. 

Stratakis CA, Chrousos GP. Capillaritis (purpura simplex) associated with use of aminoglutethimide in Cushing's 
syndrome. Am J Hosp Pharm 1994;51:2589-91. 

Stratakis CA, Chrousos GP. Endocrme tumors. In: Pizzo PA, Poplack DG, eds. Principles and practice of 
pediatric oncology. 3rd ed. Philadelphia: J.B. Lippincott, in press. 

Stratakis CA, Gold PW, Chrousos GP. Neuroendocrinology of stress: implications for growth and development, 
Horm Res 1995;43:163-7. 

Stratakis CA, Karl M, Schulte HM, Chrousos GP. Glucocorticoid resistance in humans elucidation of the 
molecular mechanisms and implications for pathophysiology, Ann NY Acad Sci 1994;746:362-76. 



4v 



ZOl HD 00618-14 DEB 

Stratakis CA, Vamvakopoulos NC, Chrousos GP. Corticotropin-releasing hormone, stress and depression. In: 
Bailey D., ed. Neuroendocrine aspects of mood disorders. Levallois, France: Laboratories Ardix, in press. 

Susman E, Dom L, Inoff-Germain G, Nottelmann E, Chrousos GP. Cortisol reactivity, distress behavior, behavior 
problems and emotionality in young adolescents, J Res Adolesc, in press. 

Tsigos C, Aral K, Latronico AC, DiGeorge A, Rapaport R, Chrousos GP. A novel mutation of the 
adrenocorticotropin receptor (ACTH-R) gene in a family with the syndrome of isolated glucocorticoid deficiency 
but no ACTH-R abnormalities in two families with triple A syndrome, J Clin Endocrinol Metab 1995;80:2186-9. 

Tsigos C, Aral K, Latronico AC, Webster E, Chrousos GP. Receptors for melanocortin peptides in the 
hypothalamic-pituitary-adrenal axis and skin, Ann NY Acad Sci, in press. 

Tsigos C, Chrousos GP. Clinical presentation, diagnosis, and treatment of Cushing's syndrome, Curr Opin 
Endocrinol Diabetes 1995;2:203-13. 

Tsigos C, Chrousos GP. The neuroendocrinology of stress. In: Krotowski D, Nosek M, Turk M, ed. Women, 
health and disability. Baltimore: Brookes Publishers, in press. 

Tsigos C, Chrousos GP. The neuroendocrinology of the stress response. In: Zachari S, ed. NIDA alcohol 
research monograph: stress, gender, and alcohol seeking behavior. Washington, DC: CSR, Inc., in press. 

Tsigos C, Chrousos GP. Physiology of the hypothalamic-pituitary-adrenal axis in health and dysregulation in 
psychiatric and autoimmune disorders, Endocrin Metab Clin N Amer 1994;23:451-66. 

Tsigos C, Chrousos GP. Stress, endocrine manifestations and diseases. In: Cooper CL, ed. Handbook of stress 
medicine. Boca Raton, FL: CRC Press, in press. 

Tsigos C, Papanicolaou D, Chrousos GP. Advances in the diagnosis and treatment of Cushing's syndrome. 
Bailliere's Clin Endocrinol Metab 1995;9:315-36. 

Vgontzas AN, Friedman TC, Chrousos GP, Bixler EO, Vela-Bueno A, Kales A. Delta sleep-inducing peptide 
in normal humans and in patients with sleep apnea and narcolepsy. Peptides, in press. 

Yanovski JA, Yanovski SZ, Harrington L, Gold P, Chrousos GP. Differences in the hypothalamic-pituitary- 
adrenal axis of black and white men, Horm Res, in press. 

Yu KC, Alexander HR, Ziessman HA, Norton JA, Buell JF, Nieman LK, Cutler GB Jr, Chrousos GP, Fraker DL. 
The role of pre-operative iodocholesterol scintiscanning in patients undergoing adrenalectomy for Cushing's 
syndrome. Surgery, in press. 



ij 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00623-12 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT 180 characters or less. Title must fit on one line between the borders.! 

Adrenal Physiology, Pathophysiology, and Molecular Biology 



PRINCIPAL INVESTIGATOR lUst other professional personnel below the Principal Investigator) (Name, title, laboratory, 

P.I.: G.B. Cutler Head 

Others: K.M. Barnes Chemist (Tech) 

G.P. Chrousos Chief, SPE, DEB 

L. Laue Adjunct Scientist 

L. Mercado-Asis Visiting Associate 

D. Merke Clinical Associate 

L. Nieman Head 

K. Oerter-Klein Special Volunter 

J. Yanovski Clinical Associate 



and institute affiliationi 

SDE, DEB, NICHD 
SDE, DEB, NICHD 
SPE, DEB, NICHD 
SPE, DEB, NICHD 
SDE, DEB, NICHD 
SDE, DEB, NICHD 
URM, DEB, NICHD 
SDE, DEB, NICHD 
SDE, DEB, NICHD 



COOPERATING UNITS lif any) 



J. Doppman, Radiology, CC; E. Oldfield, Surgical Neurology Branch, NINDS 



LAB/BRANCH 

Developmental Endocrinology Branch 



SECTION 

Section on Developmental Endocrinology 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

2.4 



PROFESSIONAL: 
2.2 



0.2 



CHECK APPROPRIATE BOX(ES) 

H (a) Human subjects D (b) Human tissues 
H (a1) Minors 
D (a2) Interviews 



n (c) Neither 



SUMMARY OF WORK {Use standard unreduced type. Do not exceed the space provided.} 

We seek to advance understanding of the mechanisms that cause adrenal androgen secretion by the fetal 
adrenal zone prenatally and by the definitive adrenal cortex during adrenarche, and to improve the diagnosis 
and treatment of disorders that cause excess adrenal androgen or glucocorticoid secretion, such as premature 
adrenarche, congenital adrenal hyperplasia, adrenal neoplasms, idiopathic hirsutism, polycystic ovary syndrome, 
and Gushing syndrome. We also seek to clarify the pathophysiology of primary adrenal insufficiency 
(Addison's disease") and secondarv adrenal insufficiency and to improve the treatment of these conditions. 

Children with congenital adrenal hyperplasia are being enrolled into a study to test the hypothesis that growth 
can be normalized, and the side effects of supraphysiologic glucocorticoid treatment avoided, by a regimen of 
antiandrogen, aromatase inhibitor, and reduced hydrocortisone dose. Additionally, the potential to cure the 21- 
hydroxylase deficiency form of congenital adrenal hyperplasia by gene therapv is being explored in a newly 
recognized animal model of this disorder. 

Patients with Cushing syndrome or a pseudo-Cushing state are being studied by several new diagnostic 
methods to determine the relative diagnostic efficiency of these new methods compared to the old. Oncogene 
expression in corticotropinomas is also under study in an attempt to elucidate the molecular basis of Cushing 
disease. 



PHS 6040 (Rev. 5/92) 



4U 



ZOl HD 00623-12 DEB 

Project Description 

Objectives: 

This project seeks to increase our understanding of adrenal physiology, pathophysiology, and molecular biology. 
The goals of the project are the study of fetal adrenal development, adrenarche, and disorders of adrenal function. 
The principal disorders under study are congenital adrenal hyperplasia, Gushing syndrome, and pseudo-Cushing 
states. 

Methods Employed: 

1) Fetal adrenal development - adrenal gland histology and steroid radioimmunoassay in newborn marmosets 
treated with substances that have been postulated to be trophic for the fetal adrenal zone. 

2) Congenital adrenal hyperplasia - steroid radioimmunoassay, evaluation of new treatments such as LHRH 
analog, antiandrogen (flutamide) and aromatase inhibitor (testolactone); adrenal transplantation and transgenic 
techniques, using a newly recognized mouse model of 21 -hydroxylase deficiency, to test the hypothesis that the 
enzyme deficiency can be cured through these approaches. 

3) Gushing syndrome and pseudo-Cushing states - evaluation of corticotropin-releasing hormone, overnight 
dexamethasone, overnight metyrapone, and combined dexamethasone/petrosal sinus catheterization as new 
approaches for the differential diagnosis of Gushing syndrome; oGRH test to predict recurrence of Gushing 
syndrome after successful microadenomectomy; oncogene expression in corticotropinomas (extraction of tumor 
mRNA and identification and analysis of suspected oncogenes by RT-PGR). 

4) Adrenal insufficiency - Steroid and peptide radioimmunoassay to study feedback regulation of 
adrenocorticotropin (AGTH) by Cortisol. 

Progress: 

1. Gongenital adrenal hyperplasia 

a. Glucocorticoid dose schedule in the treatment of GAH 

Previous studies suggested that it might be possible to develop a once-daily hydrocortisone schedule for the 
treatment of GAH. To test this hypothesis, once-daily and twice-daily schedules were compared in a double- 
blind, randomized cross-over trial of 2 years' duration (one year on each arm). Total hydrocortisone dose was 
permitted to be adjusted throughout both arms, as would be the case in clinical practice, and thus the total daily 
hydrocortisone dosage for each arm was one outcome variable of the study. Despite a 40% increase of 
hydrocortisone during the once-daily regimen, both the clinical and biochemical measures of control of GAH 
deteriorated significantly compared to the twice-daily regimen. We concluded that GAH cannot be controlled as 
effectively with a once-daily regimen, even with a substantial increase in total daily hydrocortisone dose. 

b. Supplemental salt administration in patients with GAH 

Hypovolemia due to impaired mineralocorticoid synthesis is a major stimulus to AGTH secretion in GAH. 
Although administration of fludrocortisone should in theory correct mineralocorticoid deficiency, the appropriate 
dose of fludrocortisone depends upon dietary salt intake and varies accordingly. Thus, patients on a fixed dose 
of fludrocortisone may have too much mineralocorticoid on some days and too little on others, due to fluctuations 
in dietary salt. We therefore initiated a randomized double-blind clinical trial to test the hypothesis that 
supplemental salt administration will reduce day-to-day fluctuation in AGTH secretion, and thus improve control 
of androgen secretion, by eliminating fluctuations in the hypovolemic stimulus to AGTH release. The results to 
date have indicated improved adrenal suppression, as predicted. However, the magnitude of the effect in children 
who are receiving fludrocortisone appears too small to be of clinical importance. 



5U 



ZOl HD 00623-12 DEB 

c. Treatment of CAH with a new regimen of flutamide, testolactone, and reduced hydrocortisone dose 

The successftil use of antiandrogen in familial male precocious puberty, combined with inhibition of androgen-to- 
estrogen conversion, has led us to initiate a similar approach in congenital adrenal hyperplasia. The protocol 
employs flutamide as the antiandrogen and testolactone as the inhibitor of androgen to estrogen conversion. The 
rationale for this approach is that androgen excess is an inevitable outcome of physiologic hydrocortisone dosage 
in CAH. Blockade of excess androgen offers the prospect of normal growth, whereas the conventional approach 
of suppressing excess androgen through supraphysiologic hydrocortisone dosage does not, because of the growth- 
inhibiting actions of glucocorticoid. 

Preliminary results of this new approach have recently been analyzed. Compared to conventional treatment, the 
regimen of flutamide, testolactone, and reduced hydrocortisone dose produced an increase in plasma 17- 
hydroxyprogesterone levels and a significant decline in urine Cortisol, linear growth rate, weight velocity, and 
bone maturation. No important adverse effects were observed. We conclude that the regimen of flutamide, 
testolactone, and reduced hydrocortisone dose improves the short-term control of growth and bone maturation in 
children with congenital adrenal hyperplasia. Long-term studies are required to determine whether this approach 
can normalize these children's growth and development. 

d. Testicular adrenal rest tissue in boys with congenital adrenal hyperplasia 

One of the most troublesome complications of CAH in boys is the development of testicular adrenal rest tumors. 
This complication is thought to arise because of increased ACTH levels acting on adrenal rest cells within the 
testis, an occurrence which has been described in approximately 10% of normal subjects. Thus, those boys with 
CAH who have adrenal rest cells within the testis are presumed to be at risk for this complication, whereas those 
who do not have such cells present are presumed not to be at risk. 

Although at present there is no way to determine which boys are at risk, it would be desirable to detect these 
lesions as early as possible so that attempts could be made to assure the optimal degree of ACTH suppression. 
To improve detection, Avila and his colleagues have performed a detailed analysis of the gray scale and color 
Doppler ultrasound features of the testicular adrenal rest tissues in 8 boys with this rare condition. Through 
earlier detection, we hope to develop improved methods to prevent the progression of this complication. 

2. Cushing syndrome 

a. Pathophysiology of glucocorticoid-induced growth suppression 

Previous studies in our laboratory showed that glucocorticoid-induced growth suppression could be induced in 
a single proximal tibial epiphysis by the local infusion of dexamethasone. The mechanism of this local growth 
arrest did not involve suppression of mRNA for growth hormone receptor in the affected epiphysis. Recently, 
we have used this growth suppression model to examine the hypothesis that catch-up growth after glucocorticoid 
withdrawal results from a neuroendocrine mechanism. Contrary to this hypothesis, catch-up growth after 
termination of the dexamethasone infusion was observed only in the epiphysis that had received dexamethasone. 
No change in growth rate was observed in the control contralateral epiphysis, or in the ipsilateral distal tibial 
epiphysis. Thus, catch-up growth following glucocorticoid withdrawal was a local phenomenon that occurred 
only within the affected growth plate. 

b. Differential diagnosis 

Despite recent advances in transsphenoidal surgery for Cushing disease, the remission rate for all patients 
undergoing transsphenoidal surgery has remained well below 100 percent. The causes of surgical failure include 
errors of diagnosis, including the failure to distinguish an ectopic from a pituitary source of ACTH and the failure 
to distinguish Cushing disease from pseudo-Cushing states, and inability of the surgeon to locate pituitary 
microadenomas during surgery. 



5^ 



ZOl HD 00623-12 DEB 
We have recently made the following contributions in the area of differential diagnosis: 

1) New criteria have been developed for the single-dose, overnight metyrapone test. Based on a pilot study of 
this simplified test, the diagnostic efficiency is nearly identical to that of the 2-day standard metyrapone regimen 
that we described in the Aimals of Internal Medicine in 1994. 

2) A novel combined dexamethasone/inferior petrosal sinus sampling (IPSS) test appears to improve the 
discrimination between pituitary Gushing disease and ectopic ACTH syndrome in patients with mild or 
intermittent hypercortisolism and in patients who have had recent medical treatment for hypercortisolism. 
Moreover, the absolute level of the CRH-stimulated plasma ACTH in the petrosal sinuses appears to distinguish 
patients with a pseudo-Cushing state from patients with Gushing disease. 

3) Contrary to a recent report from another medical center, cavernous sinus sampling did not lead to improved 
diagnosis compared to inferior petrosal sinus sampling. 

4) We reported the largest series to date on the diagnostic evaluation of pediatric Cushing's syndrome, showing 
that the diagnostic criteria developed previously for adults are also applicable to children and adolescents with 
this disorder. 

1) New diagnostic criteria for the single-dose overnight metyrapone test 

A pilot study involving 63 patients has been conducted to determine whether this simplified test, requiring 12 
hours rather than the 48 hours for the standard test, is as effective diagnostically as the standard test. Stimulation 
of 1 1-deoxycortisol > 225-fold, or a decrease of plasma Cortisol during metyrapone of < 45%, yielded a specificity 
of 100% and a sensitivity of 68% for the diagnosis of Gushing disease. The sensitivity of 68% was nearly 
identical to that obtained with the standard test in the same patients (67%). We conclude that the single-dose test, 
which avoids the need for timed urine collections, has nearly identical diagnostic efficiency as the standard test. 
Moreover, combining the results from the two tests increased sensitivity to 84%, which was significantly greater 
than with either test alone (p < 0.02). This illustrates the clinical value of performing additional tests when a 
single test yields negative or equivocal results. We ultimately envision that this test could be combined with two 
other noninvasive, single-dose tests (8-mg overnight dexamethasone test and the ovine corticotropin-releasing 
hormone test) to permit accurate non-invasive diagnosis of Gushing disease in nearly all subjects. 

2) Combined dexamethasone/inferior petrosal sinus sampling (DEX/IPSS) test 

One of the pitfalls of the IPSS procedure to distinguish ectopic from pituitary sources of AGTH is the patient with 
an ectopic source of AGTH who has had recent adrenal blockade or who has mild or intermittent 
hypercortisolism. The normal pituitary corticotroph cells in such patients may be unsuppressed, giving rise to 
a petrosal sinus-to-peripheral AGTH gradient that mimics the gradient seen in Gushing disease. To overcome this 
problem, we hypothesized that the administration of dexamethasone (8 mg per day for 2 days) prior to IPSS 
would fully suppress normal, but not adenomatous, corticotroph cells, leading to improved diagnosis. Preliminary 
results indicate that this is indeed the case, and that the DEX/IPSS test will improve diagnostic accuracy in this 
setting. 

3) Selective cavernous versus inferior petrosal sinus sampling for differential diagnosis of Gushing syndrome 

To test the hypothesis that cavernous sinus sampling would generate higher central-to-peripheral AGTH gradients 
and obviate the need for concurtent GRH administration to localize occult pituitary microadenomas in Gushing 
disease, we compared baseline cavernous sinus gradients to baseline and GRH-stimulated petrosal sinus gradients 
in 15 patients with surgically proven Gushing disease. Contrary to a recent report, the test sensitivity was only 
80% for the baseline cavernous sinus AGTH gradient (3 [20%] patients had a central-to-peripheral gradient <2 
and were thus false negatives) compared to 100% sensitivity for the GRH-stimulated petrosal sinus AGTH 
gradients (gradients of 26.7, 91.3, and 52.6) in the same patients. We conclude that petrosal sinus sampling with 
CRH stimulation is a more accurate diagnostic procedure than cavernous sinus sampling without GRH. 






ZOl HD 00623-12 DEB 

4) Gushing syndrome in children and adolescents 

Gushing syndrome is rare in children, and thus comprehensive information concerning diagnosis, differential 
diagnosis, and treatment are difficult to obtain. To address this need, we have analyzed the data from 59 patients 
with Gushing syndrome between the ages of 4 and 20 who were admitted to NIH during the past decade. 

Fifty patients had Gushing disease, 3 had ectopic AGTH production, and 6 had primary adrenal disease (2 with 
adrenal cancer and 4 with bilateral primary pigmented nodulocortical adrenal disease). Increased body mass index 
and decreased growth velocity were the most prominent clinical features. Pituitary microadenomas were not seen 
by MR imaging in 48% of children with Gushing disease. The high-dose dexamethasone suppression test, GRH 
test, and petrosal sinus sampling had similar diagnostic performance as in adult patients with Gushing syndrome. 
Transsphenoidal surgery was effective in 48 of 49 patients with Gushing disease. 

These data provide a comprehensive, modem approach to the diagnosis and treatment of Gushing syndrome in 
children and adolescents. They represent the largest, most accurately diagnosed, and most effectively treated 
pediatric series that has been reported thus far. 

c. Gomplications 

We have published two brief reports during the past year to alert clinicians to rare but potentially devastating 
complications of Gushing syndrome. The first report describes the initial case of spontaneous vulvar necrotizing 
fasciitis in a middle-aged women who had a small pimple-like vulvar lesion that evolved rapidly into a near-fatal 
episode requiring extensive surgical debridement, hyperbaric oxygen, intensive case, and months of recovery. 
The case illustrates the extraordinary risk posed by the immunosuppressive effect of Gushing syndrome. 

The second report describes the initial cases to appear in the literature of spontaneous hemorrhage into AGTH- 
secreting microadenomas causing Gushing disease. In each case the hemorrhages, which were not recognized 
clinically at the time of occurrence, caused temporary remission of the Gushing disease with recurrence a year 
or more afterward owing to renewed growth of the surviving tumor cells. After the hemorrhages, the biochemical 
profiles of the patients were initially consistent with secondary adrenal insufficiency, which could have led to an 
incorrect diagnosis of factitious Gushing syndrome. Thus, hemorrhage into AGTH-secreting microadenomas is 
a potential cause of unexplained remission of Gushing syndrome. 

d. Treatment 

1) Recovery from osteoporosis after cure of Gushing svndrome 

Osteoporosis is a well-recognized complication of Gushing syndrome, although relatively little is known about 
the natural history of this complication in treated patients. To gain further information about the potential of 
osteoporotic bone in Gushing syndrome to recover normal bone density after cure of hypercortisolism, patients 
with Gushing syndrome are undergoing bone density measurements by dual energy x-ray absorptiometry (DEXA) 
before and after successful treatment. If patients fail to show recovery of bone density within several years after 
cure, we plan to initiate a therapeutic trial to determine whether bone density can be improved by any of the 
treatments employed in the treatment of idiopathic or postmenopausal osteoporosis. 

2) Mechanism of hypogonadism in Gushing syndrome and effect of successfiil treatment 

Decreased plasma testosterone, impotence, and decreased libido are common in men with Gushing syndrome. 
Hypogonadism also occurs commonly in women with Gushing syndrome, but is more difficult to study because 
of the need to control for the stage of the menstrual cycle. To gain further insight into the mechanism of 
hypogonadism in Gushing syndrome, spontaneous gonadotropin secretion and the gonadotropin response to 
administration of gonadotropin-releasing hormone are being studied before and after successful treatment. 



«J •*■} 



ZOl HD 00623-12 DEB 
3) Drug treatment of ACTH-secreting tumors in Gushing disease and in Nelson syndrome 

Following bilateral adrenalectomy for Gushing disease, the pituitary tumors sometimes enlarge and secrete 
markedly increased amounts of ACTH (Nelson syndrome). Although 3 drugs have had limited effectiveness in 
suppressing the function of these tumors (cyproheptadine, bromocriptine, and valproic acid), none has achieved 
widespread clinical acceptance because of a low response rate. Since these 3 drugs are believed to act through 
different mechanisms, we conducted a study to determine whether the simultaneous use of these agents might be 
additive or synergistic in suppressing AGTH secretion in persistent Gushing disease after unsuccessful 
transsphenoidal surgery and in Nelson syndrome. With the single-dose study design that was employed, only 
bromocriptine and the combination of all three agents achieved statistically significant suppression of plasma 
AGTH. Addition of cyproheptadine and valproic acid did not have any further AGTH-suppressing effect. 

Significance: 

1) Fetal adrenal zone and adrenarche. Adrenal androgens from fetal adrenal are the major precursors for placental 
estrogen synthesis during pregnancy, which plays an important role in preparing the breast for lactation and in 
inducing uterine oxytocin receptors. Postnatally, adrenal androgens play an important pathogenetic role in 
hormone-dependent cancers such as those of the breast and prostate, and in several endocrine disorders such as 
congenital adrenal hyperplasia, premature adrenarche, hirsutism, and Gushing syndrome. Increased understanding 
of the mechanisms regulating adrenal androgens may lead to new methods of controlling adrenal androgen 
secretion in these disorders. 

2) Gongenital adrenal hyperplasia (GAH). This is among the most common autosomal recessive disorders in the 
United States population. The classic form affects approximately 1 in every 13,000 children (about the same as 
PKU), and has a heterozygote frequency of about 1 in 57. The nonclassical or late-onset form of GAH is even 
more common. Thus, advances in the treatment of this disorder will benefit the 300 children with classic GAH 
and the larger number of nonclassical patients bom annually in the United States. 

3) Hirsutism. Approximately 1 woman per 1000 (a prevalence of nearly 100,000 women in the United States) 
has hirsutism sufficiently severe to constitute a significant management problem. These women often develop 
anxieties about their feminine identity that hinder normal social development during the teen-age and adult years. 
Thus, improved understanding of the pathogenesis of this disorder, and improved treatment methods, could 
enhance the well-being of a large number of women. 

4) Gushing disease. This disorder is estimated to affect 600 to 2000 new cases per year in the United States. 
When described by Gushing in 1932, it was a devastating, uniformly fatal illness that often struck in the prime 
of life. Recent advances have greatly improved our ability to diagnose accurately and to treat effectively this 
potentially lethal disease. 

5) Adrenal insufficiency. This potentially fatal disorder continues to pose a therapeutic challenge because of the 
morbidity associated with undertreatment or overtreatment. Improved methods of treatment are needed to restore 
these patients to full health. 

Proposed Gourse: 

1) The hypothesis that adrenarche results from a proopiomelanocortin (POMG)-derived peptide will be tested by 
comparing the adrenal androgen response to corticotropin-releasing hormone (GRH), which stimulates POMG, 
to that of AGTH, which suppresses POMG. This study will be carried out in patients with hypothalamic adrenal 
insufficiency. 

2) Gongenital adrenal hyperplasia. Future studies will address the following areas: 

a) The effect on growth rate, bone maturation, predicted adult height, and final adult height of a new regimen 
consisting of antiandrogen (flutamide), aromatase inhibitor (testolactone), reduced hydrocortisone dose, and 



O'i 



ZOl HD 00623-12 DEB 



fludrocortisone versus a conventional hydrocortisone/fludrocortisone regimen. 



b) The effect, in a recently discovered strain of mice witli 21 -hydroxylase deficiency, of this disorder on the 
development of the hypothalamic CRH neuron, as determined by immunohistochemistry and in situ hybridization 
methods. 

c) Use of the 21-hydroxylase-deficient mouse to develop approaches to correct the 21 -hydroxylase deficiency by 
gene therapy approaches. 

Testing of new therapeutic strategies would benefit from the recruitment of a birth cohort of CAH patients whose 
potential adult height has not been compromised by inadequate treatment during the first 1-2 years of life. To 
identify such a cohort, we are attempting to interest the nearby state laboratories responsible for newborn 
screening in setting up a pilot newborn screening program. Effective, inexpensive methods for newborn screening 
of CAH have been developed and are in use in Italy, Japan, and the states of Washington, Alaska, Illinois, Texas 
and other states. 

3) Gushing syndrome 

To improve the usefulness of inferior petrosal sinus sampling (IPSS) in the differential diagnosis of Gushing 
disease versus ectopic AGTH secretion, additional patients will be studied with the new DEX/IPSS test. To 
decrease the time and cost of evaluation of hypercortisolism, a 1-day metyrapone test, dexamethasone test, and 
oGRH test will be evaluated prospectively. Other initiatives include efforts to define the natural history of the 
osteoporosis and hypogonadism of Gushing disease before and after successful transsphenoidal microsurgery. 
If the osteoporosis is not reversible spontaneously with cure of hypercortisolism, new therapeutic strategies will 
be developed to attempt to prevent future losses of bone density. Additionally, the effectiveness of medical 
treatment to suppress AGTH-secreting adenomas will be evaluated in Nelson syndrome and in persistent Gushing 
disease after unsuccessfiil transsphenoidal surgery. 

To gain insight into the molecular basis for the corticotroph neoplasms that cause Gushing disease, RNA from 
these neoplasms will be amplified by RT-PCR to detect the presence of potential oncogenes. Our ultimate goal 
is to gain new insights into the molecular basis for this disorder that can be used to design innovative strategies 
to eradicate these tumors or to inhibit their growth and excessive hormone secretion. 

Protocols: 

Animal 



92-016 



Yanovski Comparison of the density of hypothalamic corticotropin-releasing 

hormone neurons between normal mice and mice with congenital 
adrenal hyperplasia 



Human 



93-GH-lOl 



Merke 



Flutamide and testolactone treatment of children with classic 
congenital adrenal hyperplasia (21 -hydroxylase deficiency) 



This study tests the hypothesis that the regimen of flutamide, testolactone, and reduced hydrocortisone dose will 
normalize the growth and development of children with congenital adrenal hyperplasia (CAH). The study 
employs a randomized, open, parallel design comparing the new regimen to conventional therapy for a period of 
two years. 

The preliminary results have shown decreased growth velocity, weight velocity, and bone maturation rate during 
the new regimen despite the decrease in glucocorticoid dose and increase in the adrenal androgen precursor, 17- 
hydroxyprogesterone, in plasma. Only one child in 26 has failed to tolerate the new regimen, due to GI 



tiU 



ZOl HD 00623-12 DEB 

intolerance. No other adverse effects have been observed. We conclude that the new regimen has short-term 
effectiveness and deserves longer term study. 

90-CH-194 Yanovski Inferior petrosal sinus sampling for the determination of 

adrenocorticotropic hormone (ACTH) concentration in normal 
volunteers and in patients with disorders of the hypothalamic- 
pituitary-adrenal axis 

This study is currently testing the hypothesis that the 2-day, 8-mg dexamethasone test administered before inferior 
petrosal sinus sampling (Dex/IPSS test) will suppress normal corticotroph function during the IPSS procedure but 
will permit continued ACTH secretion from ACTH-secreting pituitary microadenomas causing Gushing disease. 
If correct, this hypothesis implies that the Dex/IPSS test will extend the diagnostic usefulness of IPSS to patients 
with intermittent Gushing syndrome, mild Gushing syndrome, or prior adrenal blockade. These are settings in 
which the conventional IPSS procedure may yield misleading results because central-to-peripheral AGTH 
gradients arising from normal corticotroph cells may be mistaken for an AGTH-secreting microadenoma. Thus, 
an erroneous diagnosis of pituitary Gushing disease may be made in the patient with ectopic AGTH syndrome 
or with an unrecognized pseudo-Gushing state. 

The study employs a randomized, open design in which the patient receives 2 IPSS procedures, one with and one 
without prior 8 mg per day dexamethasone administration for 2 days. The diagnostic performance of the 
conventional and modified IPSS procedure will then be compared against the "gold standard" diagnosis 
determined at follow-up (the "gold standard" is surgical cure or pathologic diagnosis for Gushing syndrome and 
remission or prolonged lack of progression for pseudo-Gushing states). 

Preliminary results suggest that the Dex/IPSS procedure has improved diagnostic accuracy compared to IPSS in 
settings in which normal corticofroph function may not be suppressed. We conclude that further evaluation of 
this new test is warranted. 

94-CH-0144 Asis Bone mineral density and bone metabolism in patients with unfreated 

and cured endogenous Gushing syndrome 

This study tests the hypothesis that patients with osteoporosis owing to Gushing syndrome will recover normal 
bone density within 3 years of the cure of Gushing syndrome. We fiirther hypothesize that this recovery of bone 
density will be attributable to an increased rate of bone formation without a corresponding increase in bone 
resorption. The study employs a longitudinal, observational design in which each subject's pretreatment bone 
density, bone formation markers, and bone resorption makers are compared by ANOVA for repeated measures 
with the corresponding values during the 3 years after the cure of Gushing syndrome. 

The available pretreatment results indicate low-turnover osteoporosis before treatment of Gushing syndrome, 
indicating that both the rate of bone formation and of bone resorption are suppressed. Further observation will 
be required to determine the changes following the cure of Gushing syndrome. 

PUBLIGATIONS 



Avgerinos PG, Gutler GB Jr. The Gushing syndrome (letter). Ann Intern Med 1995;I22:959. 

Avila NA, Premkumar A, Shawker TH, Jones JV, Laue L, Gutler GB Jr. Testicular adrenal rest tissue in 
congenital adrenal hyperplasia: gray scale and color Doppler ultrasound. Radiology, in press. 

Baron J, Oerter KE, Golli MJ, Yanovski JA, Novosad JA, Bacher JD, Cutler GB Jr. Catch-up growth after 
glucocorticoid excess; a mechanism intrinsic to the growth plate. Endocrinology 1994;135:1367-71. 

Cunningham DS, Gutler GB Jr. Spontaneous vulvar necrotizing fasciitis in Gushing' s syndrome, So Med 
1994;87:837-8. 



5y 



ZOl HD 00623-12 DEB 

Cutler GB Jr. Ambiguous genitalia. In: Hurst JW ed. Medicine for the practicing physician, 4th ed. Philadelphia: 
W.B. Saunders, in press. 

Doppman JL, Nieman LK, Chang R, Yanovski JA, Cutler GB Jr, Chrousos GP, Oldfield EH. Selective venous 
sampling from the cavernous sinuses is not a more reliable technique than sampling from the inferior petrosal 
sinuses in Gushing syndrome, J Clin Endocrinol Metab 1995;80:2485-89. 

Klein KG, Cutler GB Jr. The hypothalamus and neuroendocrine disorders. In: Berg BO, ed. Principles of child 
neurology. New York: McGraw-Hill, in press. 

Laue L, Cutler GB Jr. The adrenal cortex. In: Bittar EE, Bittar N, eds. Principles of medical biology. Greenwich: 
JAl Press, in press. 

Magiakou MA, Mastorakos G, Oldfield EH, Gomez MT, Doppman JL, Cutler GB Jr, Nieman LK, Chrousos GP. 
Gushing syndrome in children and adolescents: Presentation, diagnosis and therapy. N Engl J Med 1994;33 1:629- 
36. 

Mercado-Asis LB, Oldfield EH, Cutler GB Jr. Pituitary tumor hemorrhage in Gushing disease. Ann Intern Med 
1995;122:189-90. 

Nieman LK, Cutler GB Jr. Cushing's syndrome. In: DeGroot LJ ed. Endocrinology, 3rd ed. Philadelphia: W.B. 
Saunders, in press. 

White PC, Pescovitz OH, Cutler GB Jr. Synthesis and metabolism of corticosteroids. In: Becker KL, ed. 
Principles and practice of endocrinology and metabolism, 2nd edition. New York: J.B. Lippincott, in press. 

Yanovski JA, Cutler GB Jr. Glucocorticoid action and the clinical features of Gushing syndrome. In: Aron DC, 
Tyrrell JB, eds. Endocrine and metabolism clinics of North America. Philadelphia: W.B. Saunders, 1994;487- 
509. 



'Oi 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD-00627-06 DEB 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or /ess. Title must fit on one line between the borders.! 

Glycoprotein Hormones: Oligosaccharide Structure and Function 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

P.I. D. Blithe Expert UG, SME, DEB, NICHD 



Others: 



M. Nemansky 
E. Moy 
C. Lyons 



Visiting Fellow 
Clinical Associate 
Technician 



UG, SME, DEB, NICHD 
UG, SME, DEB, NICHD 
UG, SME, DEB, NICHD 



COOPERATING UNITS lif any) 

R. lies, St. Bartholomew's Hospital Medical College, West Smithfield, London, U.K.; V. Reinhold, Boston 
University School of Medicine, Boston, MA 



LAB/BRANCH 



Developmental Endocrinology Branch 



SECTION 

Unit of Glycobiology 



INSTITUTE AND LOCATION 

National Institute of Child Health and Human Development 



TOTAL STAFF YEARS: 

3.0 



PROFESSIONAL: 

3.0 



OTHER: 

0.5 



CHECK APPROPRIATE BOX(ES) 

El (a) Human subjects H (b) Human tissues 
D (a1) Minors 
D (a2) Interviews 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The goal of the project is to increase our understanding of the structure-function relationships and glycobiology of 
the oligosaccharide moieties on glycoprotein hormones. Currently under investigation are, human chorionic 
gonadotropin (hCG), gonadotropin B-core fragment, glycoprotein hormone free a subunits associated with pregnancy 
or malignancy, and pituitary free a subunits secreted throughout the normal menstrual cycle. We have shown that 
the free a subunit purified from pregnancy urine stimulates secretion of prolactin from primary cultures of human 
decidual cells in a dose-dependent manner. This finding indicated that free a is a glycoprotein hormone with 
potential functions that are independent of heterodimeric gonadotropins. We further found that free a can play a role 
in recruitment of endometrial stromal cells for decidualization. This observation expands the function of the free a 
subunit into areas of reproductive physiology that occur during the luteal phase of the menstrual cycle and may 
suggest involvement of free a in implantation. 

Carbohydrate modifications, resulting in a variety of branched glycan structures, occur on all glycoproteins prior 
to secretion. These modifications can affect virtually every aspect of the molecule's behavior, including receptor 
binding and signal fransduction, yet the underlying regulatory mechanisms remain elusive. We have investigated 
fimctional aspects of the glycan moieties on free a, demonstrating that specific types of carbohydrate modifications 
on free a can either prevent or facilitate combination with hCG-P to form intact hormone. We have found that the 
free a subunits that are located in fetal compartments in high concentrations during early pregnancy exhibit physical 
properties that are similar to those of the free a subunit isolated from pregnancy urine. We have shown that the 
oligosaccharide moieties on this early pregnancy free a function to maintain the molecule in an uncombined form in 
placental compartments that also contain high amounts of combinable free P-subunit. 

We have identified differences in the oligosaccharide moieties of free a and hCG as a function of gestational 
development: in particular, the glycosylation of free a changes dramatically as pregnancy progresses. In five 
pregnancies examined thus far, the glycosylation changes were found to begin at week 14-15 of gestation and were 
completed by week 17, suggesting developmental regulation of glycoprocessing enzymes in the cells that secrete a 
subunit. We plan to examine how the differences observed in glycosylation affect the bioactivity of these molecules. 



PHS 6040 (Rev. 5/92) 



ZOl HD00627-06 DEB 



Project Description 

Objectives: 

The overall objective of the project is to flirther our understanding of the structural elements of glycoprotein 
hormones and how these elements, particularly the carbohydrate moieties, contribute to the ftinction of these 
molecules. The molecules under study were, human chorionic gonadotropin (hCG), gonadotropin p-core fragment 
and glycoprotein hormone free a subunit. 

Glycoprotein hormone free a subunit is a major placental secretory product during pregnancy. Free a is produced 
throughout pregnancy by trophoblast cells of the placenta. Although the highest maternal serum levels of free 
a occur during third trimester, much higher concentrations of free a are observed in the extraembryonic coelomic 
fluid and the amniotic fluid in early pregnancy. Free a subunits are also secreted by the pituitary throughout the 
normal menstrual cycle in nonpregnant individuals. Recent structural data indicates that a subunit belongs to a 
superfamily of growth factors that share structural homology. We have recently described a potential function for 
free a subunit in pregnancy, demonstrating that it can stimulate secretion of prolactin from primary cultures of 
human decidual cells. In view of our findings, we propose that free a is a glycoprotein hormone with activity 
that is independent of the heterodimeric hormones (hCG, LH, FSH and TSH) that contain a subunit in 
combination with a p subunit. The hypothesis that free a is an independent glycoprotein hormone defines a new 
area of basic and clinical research on the functions, physiology, and glycobiology of free a molecules. 

In view of the dominant role that glycosylation can play in regulating hormonal activity, our goal is to elucidate 
the structure and frinction of the carbohydrate moieties on pregnancy-related hormones. Changes in glycosylation 
of these molecules were examined as a function of gestational development. Knowledge of the oligosaccharide 
structures on hCG and free a is critical for determining the contribution of specific oligosaccharides to the 
functions of these molecules. Also, knowledge of the normal structures of these molecules throughout pregnancy 
is expected to provide information about the regulation of glycosylation during pregnancy and to provide a basis 
for distinguishing aberrant structures in diseases of pregnancy. 

Methods Emploved: 

Studies on hCG and free a molecules that were secreted at different times during pregnancy were performed on 
urine samples collected at weekly intervals throughout five individual pregnancies. Exfraembryonic coelomic 
fluid (EECF) was collected via amniocentesis at weeks 7-12 of pregnancy from sixteen volunteers (collection 
performed at St. Bartholomew's Hospital, London, U.K.). Amounts of hCG, free a, hCG-P subunit, P-core 
fragment, human prolactin, and IGFBP-1 were assessed by radioimmunoassay using polyclonal or monoclonal 
antisera. In some cases, purification of hCG from free a was not desirable, therefore, we used monoclonal 
antibodies in a radioimmunoassay that recognized either intact hCG or free a with minimal cross reactivity. For 
structural characterization of hCG and free a, the hormones were purified by gel filtration and immunoaffinity 
chromatography. Carbohydrate structure was determined by lectin affinity chromatography using lectins with a 
variety of carbohydrate specificities. The lectins used in the current study were Concanavalin A, Lens culinaris, 
and Datura stramonium. Carbohydrate composition was determined after acid hydrolysis of purified material. 
Monosaccharides were separated by high pH-anion exchange HPLC and were quantified by pulsed amperomefric 
detection. 

For the free a bioassay, primary cell cultures of endomefrial stromal cells were established from endometrial 
biopsies obtained during various times of the menstrual cycle. Cells were incubated in culture media containing 
a variety of hormonal additions including the presence or absence of progesterone and various doses of a 
subunit. 

A variety of standard biochemical methods were also employed in these studies, including enzymatic digestion, 
density gradient centrifugation, SDS-polyacrylamide gel elecfrophoresis, immunoblotting, etc. 



SO 



ZOl HD00627-06 DEB 



Major Findings: 

We have recently postulated a novel function for free a in pregnancy. We have shown that the free a molecule 
purified from pregnancy urine stimulates secretion of prolactin from primary cultures of human decidual cells 
isolated from term pregnancies. The concentration of free a that stimulated prolactin was well-within the 
physiologic maternal serum free a levels during pregnancy. Thus, placental free a molecules appear to have a 
fiinctional role in pregnancy by stimulating decidual prolactin secretion, thereby identifying free a as an 
independent glycoprotein hormone. 

We have now extended our findings to show that free a is likely to play an important role in the normal 
menstrual cycle and in very early pregnancy. In addition to the fact that free a is a major placental product 
during pregnancy, free a molecules are also secreted by the pituitary throughout the normal menstrual cycle in 
a pulsatile fashion that is regulated by GnRH. Using cells obtained by endometrial biopsy, we have found that 
free a can stimulate endometrial sfromal cells to undergo cellular differentiation to become decidualized cells. 
Decidualized endomefrium is characterized by morphologic changes and by secretion of prolactin and IGFBP-1, 
and the process of decidualization is crucial for development of a uterine lining that is receptive for implantation. 
Addition of free a in a dose range that is within normal physiologic levels of the menstrual cycle results in 
morphologic changes associated with decidualization with concomitant stimulation of prolactin and IGFBP-1 
secretion. The effects of free a are dramatically enhanced by simultaneous addition of progesterone, indicated 
a synergistic response of the combined hormones compared to the additive effects of either hormone alone. These 
findings demonstrate that free a is likely to be playing a role in the preparation of the uterus for receiving the 
developing embryo. This novel concept may have important implications regarding infertility, recurrent 
spontaneous abortion and assisted reproduction. 

We examined whether hCG could produce a stimulatory effect that was similar to the activity observed with free 
a subunit. At equivalent molar concentrations, intact hCG was unable to stimulate endometrial differentiation 
either alone or in combination with progesterone. At higher doses of hCG, some stimulation was observed, 
however, during incubation of hCG with the cells, uncombined a subunit was generated from the hCG 
preparation. The amount of stimulation that was observed with high concenfrations of hCG could be attributed 
to the uncombined a subunit that was generated during the incubation period. The observation that uncombined 
a subunit can be generated from hCG preparations and that this a subunit is bioactive may be important in 
designing optimal hormonal conditions for use in assisted reproduction protocols. 

Exfraembryonic coelomic fluid (EECF) occupies the space between the amniotic membrane and the chorionic 
membrane of the developing trophoblast representing a prominent cavity until about 12 weeks of pregnancy. The 
molar concentration of free a in the EECF is the highest level ever reported for a physiologic compartment, far 
exceeding the levels in amniotic fluid or maternal serum at the same point of gestational development. In addition 
to free a, this cavity also contains high concenfrations of intact hCG and free (3 subunit in a molar ratio of 6:3:1, 
respectively. We have shown previously that modifications to the N-linked glycans of free a occur during 
glycoprocessing of the molecule resulting in branched glycan structures that can prevent free a from combining 
with hCGp-subunit. To determine if a similar phenomenon occurred in the placentally-derived subunits of the 
EECF, we examined the EECF free a and free P populations for their abilities to combine with the 
complementary dissociated subunit. We showed that the EECF free P was fully capable of combination, whereas 
nearly all of the EECF free a subunits were unable to combine. However, when we treated the EECF free a with 
N-glycanase to remove the N-linked oligosaccharides, we could convert the bulk of the free a molecules into 
combinable subunits. Therefore, we were able to conclude that the N-linked glycans on free a protect it from 
combining with the available and combinable free P-subunits that coexist in the same placental compartment. 
Thus, this single modification to the a subunit can protect free populations of both a and P subunits. 

We have purified hCG and free a from the EECF and have demonsfrated that EECF-free a is larger than the a- 
subunit found in EECF-hCG. The larger size of the EECF-free a suggests that it is a secreted molecule rather 
than a product of dissociation or degradation of hCG. We have analyzed the carbohydrate composition of the 
EECF free a and found that it very closely resembles the overall composition of the free a purified from 



GU 



ZOl HD00627-06 DEB 



pregnancy urine. However, there appear to be substantial proteolytic cleavages in the free a obtained from urine 
as evidenced by smaller molecular size and a heterogeneity of bands of the reduced material on SDS-PAGE. 
Furthermore, treatment of the urinary free a with N-glycanase to remove oligosaccharides did not yield an 
increase in combinable forms of a, suggesting that peptide components necessary for combination may have been 
damaged or removed in the process of clearance into the urine. 

The glycosylation of hCG and free a had been assumed to be unchanged throughout pregnancy, however, we 
have shown that their glycosylation does change as gestation advances. We have examined the time points 
between week 12 and week 28, previously designated as early and late pregnancy, respectively, to determine if 
the changes in glycosylation were gradual throughout gestation, or occurred suddenly at a specific point in 
gestational development. Particularly on free a, and to a lesser extent on hCG, changes in the branching and 
fucosylation of the glycans were observed as pregnancy progressed. The glycosylation patterns between 
individual healthy pregnancies were remarkably similar to one another when the same period of gestation was 
compared, and all of the individuals displayed similar changes in the glycosylation of free a within a narrow 
time-frame of early second trimester. It is important to note that similar changes in glycosylation are also 
observed after malignant transformation, and that alterations in the glycosylation of hCG and a-fetoprotein have 
been suggested as potential diagnostic markers for malignancy. However, normative patterns for hCG and free 
a glycosylation throughout uncomplicated pregnancies have not been established. Since there are extensive 
parallels between malignant transformation and embryonic development, it is crucial to obtain these normative 
data in order to establish aberrant glycosylation profiles in disease. In each of the five individual pregnancies 
we have determined that the bulk of the change occurred between weeks 14 and 16. After reaching the 
glycosylation pattern achieved by week 1 7, the profile remained constant for the remainder of the pregnancy. 
It is likely that the changes in the glycosylation patterns reflect alterations in some key glycoprocessing enzymes 
that are under developmental regulation. 

Previously, we had developed a highly specific assay for measuring the p-core fragment of hCG in the presence 
of structurally similar molecules such as P-subunit and intact hCG. Elevated levels of P-core had been observed 
in a number of malignancies and we had postulated that our assay might have clinical utility in identification of 
abnormal conditions. Recently, lies and colleagues had observed that p-core levels were elevated in pregnancies 
affected by Down's Syndrome, however, their initial observations were limited to a small number of samples. 
We are currently participating in a multi-center study to evaluate the potential utility of gonadotropin P-core 
fragment as a marker of pregnancy affected by Down's Syndrome. In a blinded study of 373 samples, using a 
cutoff of 95% we were able identify 13 of 22 (64%) pregnancies affected with Down's Syndrome using our P- 
core assay. If this detection rate can be sustained in a larger prospective study, P-core would be the best single 
biochemical indicator currently available. Measurement of P-core is performed as a non-invasive urine test and 
the results could potentially be combined with other indicators to yield a very high rate of detection that would 
identify a population that could undergo amniocentesis to confirm the diagnosis. The abilify to detect the elevated 
P-core levels associated with Down's Syndrome did not appear to be affected by maternal age. Thus, this non- 
invasive test may be useful as a screening device for pregnancies that would be considered at low risk for the 
chromosomal abnormality. 

Significance to Biomedical Research and the Program of the Institute: 

In spite of the copious production of gonadotropin free a subunit in pregnancy, no biological role had been 
established for it. We have now identified two important activities of free a. Our earlier studies demonstrated 
that free a could stimulate prolactin secretion from terminally differentiated decidual cells suggesting that 
placental free a frinctions in the maintenance of an established pregnancy. Our most recent experiments indicate 
that free a can work synergistically with progesterone to induce decidualization of uterine endometrial stromal 
cells in culture. These results imply that free a secreted by the pituitary plays a role in preparation of the uterus 
for implantation. Thus, free a is a glycoprotein hormone with bioactivity that occurs during the normal menstrual 
cycle and it is likely that free a also has endocrine and paracrine fiinctions during very early pregnancy when 
it is being secreted by trophoblast cells of the developing placenta. This novel concept will require expansion 
of the scope of endocrinological investigations to include potential contributions from free a, in addition to those 



6 



ZOl HD00627-06 DEB 



of the heterodimeric members of the gonadotropin family. Elucidation of the functions of free a may have 
important implications for certain cases of infertility, luteal phase defects, or recurrent miscarriage. Furthermore, 
closer monitoring of the free a subunit levels in procedures used for assisted reproduction may be necessary. It 
is conceivable that free a may have a role in implantation such that too much or too little free a could be 
undesirable. Additionally, our discovery that free a acts synergistically with progesterone has important 
implications for timing events that might control the implantation window. 

We previously had compared the carbohydrate structures on the free a molecule in pregnancy with those on the 
hCG-a subunit isolated after dissociation of intact hCG. Although the two a molecules contain the same 
polypeptide structure, we showed that they have distinctly different carbohydrate moieties and that these 
carbohydrate structures could either prohibit or facilitate combination with hCG-p subunit to form intact hCG. 
In normal pregnancy, the vast majority of a polypeptides that are synthesized during the second and third 
trimesters of pregnancy become free a molecules. The recent finding that in the first trimester, the EECF 
compartment contains amounts of free a that are higher than those of hCG on a molar basis suggests that there 
may be a role for free a in early pregnancy. Our finding that this EECF-free a is larger than the a subunit 
contained within the intact EECF-hCG molecule implies that, like the free a that we have isolated from pregnancy 
urine, EECF-free a is synthesized with carbohydrate modifications that prevent combination with P-subunit. 
Since P-subunit is also present in substantial amounts in the EECF, it may be more crucial to prevent post- 
secretory combination of a and p in the EECF or amniotic fluid than in maternal serum where P-subunit 
represents a very minor component. The larger size of the EECF-free a also strongly argues against the 
hypothesis that the free subunits could originate from dissociation of nicked forms of hCG. 

The glycosylation of these molecules had been assumed to be unchanged throughout pregnancy, however, we have 
now shown that glycosylation changes dramatically as gestation advances. We have established that this change 
in the glycosylation profile of free a occurs during a relatively narrow window of gestation, between weeks 14 
and 17 of pregnancy. Having demonstrated that there are developmentally associated changes in the glycosylation 
of free a, it may be possible to determine the specific glycoprocessing enzymes involved and to identify the 
regulatory elements for those enzymes. 

It is important to note that similar changes in glycosylation branching and flicosylation are also observed after 
malignant transformation, and alterations in glycosylation of hCG and a-fetoprotein have been suggested as 
potential diagnostic markers for malignancy. However, normative patterns for glycosylation of these molecules 
throughout uncomplicated pregnancies have not been established. Since there are extensive parallels between 
malignant transformation and embryonic development, it is crucial to obtain these normative data in order to 
establish aberrant glycosylation profiles in disease. 

We have previously developed a highly specific assay for measuring the p-core fragment of hCG in the presence 
of structurally similar molecules such as P-subunit and intact hCG. Recently, lies and colleagues had suggested 
that P-core levels might be elevated in pregnancies affected by Down's Syndrome, however, their initial 
observations were limited to a small number of samples. We are currently participating in a multi-center study 
to evaluate the potential utility of gonadotropin P-core fragment as a marker of pregnancy affected by Down's 
Syndrome. Using our P-core assay, we were able to detect 64% of Down's-affected pregnancies with a false 
positive rate of 5%. If this detection rate can be sustained in a larger prospective study, P-core would be the best 
single predictor currently available. Measurement of P-core is performed as a non-invasive urine test and the 
results could potentially be combined with other indicators to yield a very high rate of detection that would 
identify a population that could undergo the more invasive techniques to confirm the diagnosis. This screening 
test has the potential to diagnose Down's-affected pregnancies regardless of maternal age. 

Proposed Research 

We have described preliminary findings indicating that free a is likely to be a glycoprotein hormone with activity 
that is independent of hCG. This novel concept will require expansion of the scope of endocrinological 
investigations to include potential contributions from free a, in addition to those of the other members of the 



f^9 



ZOl HD00627-06 DEB 



gonadotropin family. The goal of the current research is to elucidate the function and glycobiology of free a in 
reproduction and development. The specific aims are divided into three main areas: A) To investigate the 
hormonal ftinctions of free a, in particular, to define the role of free a in the regulation of human decidual 
function in pregnancy, in cellular differentiation in the normal menstrual cycle and in cellular differentiation 
during fetal pituitary development; B) To elucidate the structure and function of carbohydrate moieties on free 
a and hCG, and to ascertain the nature and the role of changes in the glycosylation of these molecules that occur 
as a function of gestational age; C) to evaluate the clinical utility of gonadotropin P-core fragment as a marker 
for clinical problems, in particular, as a screen for Down's syndrome or for early detection of recurrence of 
malignancy. 

We will pursue studies to elucidate the function of free a as an independent hormone. Having demonstrated that 
free a evokes a response from decidual cells, we plan to show that the response is a receptor-mediated 
phenomenon. We will perform binding studies on ceils obtained from endometrial biopsies to characterize the 
putative receptor. We will investigate the potential functions of free a in fetal pituitary lactotrope differentiation 
using a recently developed transgenic mouse lacking a subunit. 

We are continuing our structural characterization of the changes that occur on hCG and free a obtained from early 
and late pregnancy. We have identified a narrow gestational window in which the glycosylation changes occur. 
We will use sensitive mass spectrometry techniques to determine the explicit structural changes that are occurring 
during this time period. These structures will pinpoint specific enzymes which may be targets of developmental 
regulation. We will determine if the developmental changes in the glycosylation of free a are found at both 
glycosylation sites or predominate at a single site. Also, having demonstrated that there are dramatic structural 
differences on free a between early and late pregnancy, it is important to examine how these structures contribute 
to the various bioactivities of free a. 

We will continue to participate in a multicenter study to evaluate the potential clinical utility of P-core fragment 
levels as a marker for Down's syndrome in pregnancy. We will also screen patients who have had surgery to 
remove hCG-producing tumors, to determine if urinary p-core levels might detect recurrence of a malignancy 
prior to observations of hCG in serum. 

Protocols: 

93-CH-0075 Blithe The role of gonadotropin free a subunit in the regulation of decidual 

cell function. 

The goal of this protocol is to investigate the hormonal function of gonadotropin free a subunit, in particular, to 
define the role of free a in the regulation of human decidual function. We are exploring a possible role for 
gonadofropin free a subunit in the differentiation of endometrial stromal cells obtained from endomefrial biopsies 
performed at mid-cycle when decidualization may begin to occur. 

B-93-009(NNMC) Nash The role of glycoprotein hormone alpha subunit in the regulation of 

human decidual function in pregnancy. 

The goal of this protocol is to investigate the hormonal function of gonadotropin free a subunit in pregnancy, 
in particular, to define the role of free a in the regulation of human decidual function. We are exploring a 
possible role for gonadotropin free a subunit in stimulation of prolactin secretion from decidual cells obtained 
from term pregnancy placental membranes. 

Publications: 

Blithe DL, lies RK. The role of glycosylation in regulating glycoprotein hormone free alpha and free beta subunit 
combination in the exfra-embryonic coelomic fluid of early pregnancy, Endocrinology, 1995;136:903-10. 



6 



VJ 



ZOl HD00627-06 DEB 



Kraiem Z, Sadeh O, Blithe DL, Nisula BC. Human chorionic gonadotropin stimulates hormone secretion, iodide 
uptake, organification and cAMP formation in cultured human thyrocytes, J Clin Endocrinol Metab, 1994;79;595- 
9. 

Thotokura NR, Blithe DL Glycoprotein hormones: glycobiology of gonadotropins, thyrotropin and free alpha 
subunit, Glycobiology 1995;5:3-10. 

Patents: 

Blithe DL, Wehmann RE, Nisula BC. U.S. Patent 5, 445, 968: Purification of human chorionic gonadotropin P- 
core molecule and preparation of antibodies with specificity for same, August 29, 1995. A product from this 
application, a P-core detection kit, has been licensed and marketed by Triton Laboratories, a division of Ciba 
Coming Diagnostics Corporation. 

U.S. Serial No. 08-448,079; Blithe, D.L., Wehmann, R.E. and Nisula, B.C. Purification of human chorionic 
gonadotropin p-core molecule and preparation of antibodies with specificity for same. Continuation-in-part of U.S. 
Serial No. 07-292,985. Foreign filing license granted. Patent pending. 



fl 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00628-06 DEB 



PERIOD COVERED 

October 1, 1994 throush September 30, 1995 



TITLE OF PROJECT (80 characters or /ess. Title must fit on one line between the borders.) 

Biological roles and mechanisms of action of insulin-like growth factors (IGFs) 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) IName, title, laboratory, and institute affiliation) 

P.I. C.A. Bondy Senior Clinical Investigator SGM, DEB, NICHD 



Others: 



O. Adesanya 
E. Chin 
C. Mitchell 
R. Reinhardt 
E. Wang 
J- Wang 



Clinical Associate 
Senior Staff Fellow 
Pre-IRTA Fellow 
Clinical Associate 
Senior Staff Fellow 
Special Volunteer 



SGM, DEB, NICHD 
SGM, DEB, NICHD 
SGM, DEB, NICHD 
SGM, DEB, NICHD 
SGM, DEB, NICHD 
SGM. DF-R. NICHD 



COOPERATING UNITS lif any) 

None 



LAB/BRANCH 



Developmental Endocrinology Branch 



SECTION 

Section on Growth and Metabolism 



INSTITUTE AND LOCATION 

National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

6.0 



PROFESSIONAL: 

5.0 



OTHER: 

1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



H (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This Section carries out research on the biological role and mechanisms of action of insulin-like growth factors -I 
and -II (IGFs-I and -II). These particular growth factors have major roles in development as proven by the fact that 
targeted deletion of IGF-I and IGF-II gene expression results in severe dwarfism and, in the case of IGF-I, 90% 
mortality — although how IGF deletion causes these defects is not yet understood. Molecular, immunological and 
radioligand probes are used to analyze the cellular sites of synthesis and action of these growth factors, their putative 
regulators and potential targets during embryonic development as well as in different physiological and 
pathophysiological situations in which IGFs have key roles. 

This Section has made progress over the past year in dissecting the signal transduction pathways and biological 
functions of IGFs in the brain. A specific protease termed IDE has been implicated in selective termination of IGF 
induced signalling and two distinct cGMP-inhibited phosphodiesterase isoforms (PDE3A and PDE3B) have been 
implicated in IGF signal transmission in the developing and mature brain, respectively. Spatiotemporal patterns of 
IGF system expression have been docummented in the developing chick brain, establishing the fimdamental 
conservation of expression patterns and probable roles in avian and mammalian species. Additional studies have 
implicated specific IGF system responses in recovery from acute and chronic ischemia and a variety of demyelinating 
diatheses. 

The group has defined the mechanism by which IGF-I promotes ovarian follicluar selection, showing that this 
intrinsically produced peptide stimulates granulosa cell division and FSH receptor expression, thus regulating 
gonadotropin responsiveness and ultimately estrogen production and ovulation. A non-human primate model is 
being developed to study the effects of different menopausal hormone replacement therapy regimens on local growth 
factor production and tissue hyperplasia in female reproductive organs. A major new finding is that the addition of 
progesterone to estrogen treatment produces a drastic upregulation of IGF-1 in the primate myometrium which is 
strongly correlated with myometrial proliferation. These data are predicted to have major impact with regard to the 
design of hormone replacement therapy regimen for women at risk for myometrial tumors and for treatment of 
leiomyomas. 



65 



PHS 6040 (Rev. 5/92) 



HD-00628-06 DEB 



PROJECT DESCRIPTION: 



Objectives/Methods: 



The goal of this project is to elucidate the biological roles and mechanisms of action of insulin-like growth 
factors-I and -II (IGF-I and -II). A very fruitfiil approach to unfolding the IGF story has been investigation of 
the the microenvironmental context in which the peptides, the different receptors and binding proteins are 
expressed in vivo, both during the course of normal development and in response to experimentally induced and 
naturally occuring perturbations. Information about the cell types expressing the different components of the IGF 
system, physiological changes in levels of gene expression in these different cell types and the nature of cellular 
functions associated with IGF expression provides insight into IGF's physiological roles in a number of different 
settings. In vitro studies have identified putative regulators of IGF expression and molecular components 
implicated in IGF-induced signal transduction and these entities are investigated with respect to their in vivo 
involvement in IGF system expression and fucntion. The ultimate goal is to apply knowledge of IGF's biological 
roles in the diagnosis and treatment of human developmental and growth disorders and disease. 

Major findings: 

1) Molecular mechanisms of IGF action in the nervous system 

a) Relationship to insulin degrading enzyme and cGMP-inhibited phosphodiesterases. 

Insulin-degrading enzyme (IDE) is a serine protease linked specifically to the degradation and hence 
signal termination for insulin. To determine if IDE could also be instrumental in proteolysis and termination of 
IGF signaling in the CNS, we compared the ontogeny and neuroanatomical expression at the cellular level of IDE 
and the IGF-I/II receptor and cognate ligands. There is in fact a striking correlation between IDE and IGF-I 
receptor expression at all stages of development, suggesting that following internalization of the peptide-receptor 
complex, IDE-catalyzed degradation of the peptide is a mechanism to regulate the duration of IGF action. 

Further studies were aimed at the elucidation of signal transduction pathways of IGFs in the CNS. Cyclic GMP- 
inhibited phosphodiesterases are important regulators of intracellular cAMP levels and are uniquely sensitive to 
inhibition by cGMP. They are also distinct from other phosphodiesterase species in that they are SELECTIVELY 
REGULATED BY INSULFN. This phosphodiesterase family contains two different isoforms (PDE3A & 3B) 
encoded by distinct genes and serving tissue-specific roles in regulation of lipolysis, glycogenolysis, myocardial 
contractility, and smooth muscle relaxation. IGF-I has been shown to stimulate PDE3 activity leading to a 
significant reduction in cAMP levels in various cell types, suggesting that this class of enzyme is important in 
IGF-mediated signal transduction. To determine if one or both of these isoforms could be involved in IGF signal 
transmission in brain, we compared cellular patterns of PDE3 A&B with IGF system expression in the developing 
and mature rat brain. The two cGMP-inhibited phosphodiesterase isoforms show distinctive patterns of gene 
expression; PDE3B expression is similar to the IGF-I receptor while PDE3A expression demonstrates a high level 
of spatiotemporal heterogeneity and parallelism with local IGF-I expression, suggesting that 
this isoform is more highly regulated and subserves a variety of developmental stage- and system-specific 
functions. 

The striking parallelism between PDE3A and IGF-I expression and between PDE3B and IGF-I receptor expression 
supports the view that these enzymes are involved in IGF signal transduction in the developing and mature brain. 
In general, the high degree of relevance to IGFs noted for molecules originally identified as mediators of insulin 
action support the view that IGFs and insulin act in fundmenta lly similar ways. 



6u 



HD-00628-06 DEB 



b) IGFs - comparative neuroanatomy 

To determine if our hypotheses derived primarily from the study of murine species concerning the 
fundamental role of the IGF system in brain development were more broadly relevant across very different 
species, we investigated cellualr patterns of IGF system gene expression in the developing chick CNS. Specific 
cDNAs encoding chicken IGF-I and insulin and cognate receptors were obtained and used to generate probes for 
in situ and solution hybridization analysis of brain and retinal development from E2 to E20. Patterns of 
expression for both receptors were found to be identical to those we previously documented in the rat. IGF-II 
is concentrated in brain vasculature, meninges and chorid plexus, as in all other species and is also concnetrated 
in projection neurons during a late stage of neuronal development as i have previously described for IGF-I in the 
rat. Heterologous probes for IGFBP2 and 5 were also used revealing patterns of neuroepithelial expression for 
these two modulators of IGF action similar or identical to those seen in the rodent. Thus, it appears that IGF 
system may play a similar role in CNS development in diverse murine and avian species, supporting the view 
of a highly conserved, fundamental role for IGFs in brain development. 

c) IGFs in CNS injury responses 

We first demonstrated that there is a strong induction of IGF system expression in response to acute 
ischemia and also in the local responses to demyelinating insults several years ago. Subequently, in collaboration 
with a number of other groups, we have further delineated the time course and cell-type specificity of these IGF 
system responses to neural injury. With regard to IGF system involvement in recovery from demyelinating 
insults, important new findings indicate that astrocytic production of IGFBP2 together with IGF-I is necessary 
for mylein regeneration. 

d) IGFs cross the blood brain barrier 

In vivo intra-carotid artery infiisions were employed to compare amounts and targets of radiolabelled 
IGF-I, -II and insulin crossing the blood brain barrier (BBB). Results suggest that IGF-I and -II cross the BBB 
significantly more readily than insulin, and that they are selectively targeted to certain brain sites, most notably 
the PVN and mediodorsal nucleus of the thalamus, while insulin is largely trapped in the cerebral vasculature. 
The mechanism by which IGF's are selectively transported into the brain parenchyma appears to involve one of 
the high-affinity IGF binding proteins, IGFBP2. 

2. IGFs in skeletogenesis 

The cellular pattern of IGF system in developing cartilage and bone was documented in the rat and mouse. 
Developing early chondroblasts identified by collagen II gene expression have high level IGF-I receptor, IGF-II 
and IGFBP5 gene expression; IGF receptor and IGF-II remain high during chondrocyte development and 
differentiation well into the postnatal period, but IGFBP5 expression is lost as chondrocytes mature. Suprisingly 
in view of previous reports, IGF-I mRN is not detected in developing cartilage. IGF-I and other system 
components are not detected in developing skeleton until the onset of ossification. IGFBP3 mRNA is highly 
abundant in sprouting capillaries invading the periosteum and eventually forming the marrow cavity and IGF-I, 
IGFBP4 and 5 mRNAs are all present in osteoblasts. In postnatal day 25 long bones, gene expression for the IGF 
system is disposed as follows: IGF-II in articular and growth plate chondrocytes and in the periosteum; IGF-I, 
IGFBP4 and 5 in osteoblasts; IGFBP3 in periosteal and marrow cavity capillary endothelium and IGFBP2 in 
scattered marrow cells, but never in cartilage or bone cells. 

These results challenge the established view that endogenous IGF-I production is critical for chondrogenesis and 
suggest, instead, that IGF-II is predominant in chondrogenesis and IGF-I in osteogenesis. In addition, our data 
indicate a transient relationship between IGFBP5 and IGF-II in an early stage of chondroblast development. 



6V 



HD-00628-06 DEB 



Finally, we provide evidence for a functional interaction between IGF-I and IGFBP 4 & 5 expression in 
osteoblasts involved in both endochondral and membranous ossification. 

3) IGFs in diabetic nephropathy 

A streptozotocin-induced model of diabetes mellitus in rats was used to investigate the potential role of the IGF 
system in generating or perpetuating diabetic nephropathy. Profound changes were seen in both gene and protein 
expression of IGF-I, IGF-I receptor and 3 of the 6 IGF binding proteins. The spatiotemporal patterns of these 
changes correlate with morphological changes in renal cortex (i.e,m thickening ofglomerula basement membrane, 
hypertrophy of proximal tubule segments) and changes in renal function (i.e., increased proteinuria), strongly 
implicating intrinsic renal IGF system dysregulation in the pathogenesis of diabetic nephropathy. Clinical 
correlation with human materials is now required to extend these exciting findings. 

4. IGFs and the reproductive system 

a) Ovary 

We have previously reported that IGF-I mRNA is restricted to granulosa cells in a subset of preovulatory 
follicles in the ovary. To elucidate the follicular correlates of IGF-I expression, we analyzed IGF-I positive 
follicles with respect to bromodeoxyuridine (BRDU) incorporation, FSH receptor (FSHR) mRNA, c-fos and c-jun 
immunoreactivity in serial sections from prepubertal and random-cycling mature rat and mouse ovaries. BRDU 
incorporation and FSHR mRNA are selectively and exclusively detected in IGF-I-positive follicles, while neither 
c-fos nor c-jun are detected in these same follicles. Spearman correlation analysis of more than 70 individual 
follicles yielded highly significant positive correlations between IGF-I and BRDU and negative correlations for 
IGF-I and c-fos/jun with p< 0.001 for both analyses; these results showed independence of pubertal or estrus cycle 
status. Both c-fos and c-jun immunoreactivities are concentrated in corpora luteal granulosa cell nuclei and c-jun 
alone is concentrated in apoptotic granulosa cell and theca-interstital cell nuclei. We also investigated IGF-I and 
FSHR expression in ovarian follicles from hypophysectomized (Hx) and hormone-replaced rats, as well as normal 
and IGF-I null mice. Follicular IGF-I and FSHR mRNA levels were unaltered in the Hx model but FSHR 
mRNA levels were below the limits of detection in the IGF-I null mouse ovary, suggesting that pituitary- 
independent follicular IGF-I production regulates granulosa cell FSHR gene expression. 

In summary: 1) There is a highly significant positive correlation between granulosa cell IGF-I and FSHR gene 
expression and follicular growth, as measured by BRDU incorporation, in the murine ovary. 2) Studies in 
prepubertal and Hx animals have excluded the possibility that FSH regulates follicular IGF-I production whereas 
data from IGF-I null mice strongly suggests that IGF-I, directly or indirectly, stimulates granulosa cell FSH 
receptor expression and thus possibly follicular growth in response to FSH. 3) Cellular patterns of fos and jun 
expression in the ovary implicate the fos/jun heterodimer in granulosa cell luteinization, while expression of c-jun 
in the absence of c-fos is implicated in programmed granulosa cell death. 

b) Uterus 

We have previously documented cellular patterns of IGF system expression in the human ovary, testis 
and endometrium and established that the rodent is not a good model for local IGF system cellular patterns of 
expression or regulation as it exists in the human. Because of the difficulty working with human tissues, we are 
developingg a non-human primate model to have better experimental control and more readily available sample 
materials. 

To investigate the role of locally produced insulin-like growth factors (IGFs) in sex steroid induced 
growth in the primate uterus, ovariectomized rhesus monkeys were freated with placebo (control), estradiol (E2) 



68 



HD-00628-06 DEB 



alone or estradiol plus progesterone (E&P). After two weeks uteri were removed and serial uterine sections were 
analyzed by in situ hybridization for IGF-I, IGF-11 and IGF-I and -II receptor mRNAs and by 
inununocytochemistry for detection of the cell proliferation antigen Ki-67. IGF-I and IGF-II and both IGF 
receptor mRNAs are co-expressed by smooth muscle cells, supporting the possibility for autocrine/paracrine IGF 
action in stimulating myometrial growth. IGF-I mRNA is barely detected in control myometrium, is significantly 
increased by E2 treatment and is augmented even more by combination E&P treatment, while little change is 
noted in IGF-II or IGF receptor mRNA levels. Ki-67 positive myometrial nuclei are also significantly increased 
by E2 and are augmented more by E&P treatment, with a correlation between local IGF-I mRNA concentration 
and local Ki-67 positive cell count of r= 0.89 (p<0.001). These data provide the first experimental evidence for 
regulation of IGF-I gene expression by sex steroids in the primate uterus and implicate local IGF-I production 
in both estrogen- and progesterone-induced myometrial growth. 

Significance to Biomedical Research and the Program of the ICD: 

Our analysis of cellular and metabolic patterns of IGF system expression in diverse systems during normal 
development and in response to a variety of physiological stimuli suggests that locally synthesized IGFs function 
as autocrine or paracrine anabolic boosters, providing — in some as yet poorly understood fashion — 
for more efficient extraction and/or utilization of energy substrates and building blocks by IGF-expressing cells. 
These cells are thus able to divide more rapidly, grow bigger or work harder than surrounding cells, with the 
specific direction of the anabolic response in each case being determined by the baseline state of the cell and local 
contextual factors. 

Proposed Course: 

The major thrust of our work over the next few years is directed at testing the validity of this view of IGFs' 
biological role and elucidating the factors which regulate IGF expression and the mechanisms by which IGFs act 
to produce their anabolic effects in vivo. Specifically: 

Tissue- and developmental stage-specific hypotheses as to IGF anabolic fiinctions will be evaluated by analyzing 
the morphological and functional consequences of IGF-I and -II deletions in the nervous, reproductive and renal 
systems of targeted deletant mice (developed by Dr. A. Efstradiatis and now being bred in our lab). 

Histological mapping studies will be expanded to investigate what specific metabolic signals might be implicated 
in inducing IGF expression and what energy-aggrandizing factors may, in turn, be induced by IGFs, and by which 
signal transduction pathways. 

A major initiative on this project over the next few years will establish a novel model system in the Rhesus 
monkey for analysis of the effects of clinically important hormonal treatments on growth characteristics and hence 
potential cancer risk of mammary and uterine tissues. The autocrine/paracrine actions of insulin-like growth 
factors (IGF-I and -II) are implicated in both normal and neoplastic growth of breast and uterus and these factors, 
at least in human cell lines, are potently regulated by sex steroids. Preliminary data show that cell-specific 
regulation of IGF expression by estrogen and progesterone contributes to the differential growth effects 
demonstrated by these sex steroids in reproductive tissues. Thus, an expanded study is proposed to fully evaluate 
the effects of estrogen, progesterone and tamoxifen on IGF system expression in mammary and uterine tissues. 
Hormonally-induced changes in the level of IGF, IGF receptor and IGF binding protein expression in specific cell 
types will be quantitated and statistically compared to the local mitotic index to assess the impact of IGF changes 
on local growth. Hormone treatments which increase IGF and IGF receptor or decrease inhibitory IGF binding 
protein expression are predicted to stimulate local hyperplasia and thus provide fertile ground for the emergence 
and support of neoplastic growth. These studies will elucidate the basic biology of growth regulation in primate 
mammary and uterine tissues and identify those hormonal treatment regimens which may predispose to cancer 



HD-00628-06 DEB 



risk thus allowing the rational development of hormone regimens designed to avoid these risks. 



Publications: 



Bach MA, Chin E, Bondy CA. The effects of recombinant insulin-like growth factor I (IGF-I) on growth 
hormone, IGF-II, IGF binding protein and blood glucose levels in normal and diabetic adolescents, JCEM 
1994;79:1040-6. 

Bondy CA, Chin E, Zhou J. Significant species differences in local IGF-I and IGF-II gene expression. In: LeRoith 
D, Raizada M, eds. Current directions in insulin-like growth factor research. New York, Raven Press, pp. 73-7. 

dela Rosa EJ, Bondy CA, Wu X, Zhou J, Scavo LM, de Pablo F. Differential gene expression of insulin and 
insulin-like growth factors in the developing chicken retina and eye, Eur J Neurosci 1994;6:1801-10. 

Flyvberg A, Landau D, Kiess W, Bondy C, Chin E, Raz I, Phillip M, leRoith D. The role of IGFs and IGF 
binding proteins in diabetic nephropathy. In: Proceedings of the 15th International Diabetes Federation Meeting 
in Kobe Japan; Elsevier Science Publications, in press. 

Gehrmann J, Yao D, Bonetti B, Bondy C, Brenner M, Zhou J, Kreutzberg GW, Webster H. Expression of insulin- 
like growth factor-I and related peptides during motoneuron regeneration, Exp Neurol 1994;128:202-10. 

Liu X, Yao DL, Bondy CA, Brenner M, Hudson L, Zhou J, Webster HdeF. Astrocytes express IGF-I and IGFBP2 
during demyelination induced by experimental autoimmune encephalomyelitis, Molec Cell Neurosci 1994:418-29. 

Landau D, Chin E, Bondy, C, Flygeberg A, Roberts CT, LeRoith D. Region-specifc changes in renal IGF system 
expression during diabetes mellitus. Endocrinology 1995;138:1835-43. 

Gehrman J, Yao D-L, Bonetti B, Brenner M, Bondy CA, Wekerle H, Kreutzberg GW, Webster, H. Astrocytes 
upregulate glial fibrillary acidic protein but not IGF-I during experimental autoimmune neuritis. Brain Path 
1995;5:1-10. 

Reinhardt RR, Bondy CA. Differential cellular pattern of gene expression for two distinct cGMP-inhibited cyclic 
nucleotide phosphodiesterases in developing and mature rat brain, Neuroscience, in press. 

Reinhardt RR, Bondy CA. Insulin-like growth factors cross the blood brain barrier. Endocrinology 1994;I35:I753- 
62. 

Reinhardt RR, Chin E, Zhou J, Taira M, Murata T, Manganiello VC, Bondy CA. Distinctive anatomical patterns 
of gene expression for cGMP-inhibited cyclic nucleotide phosphodiesterase subfamilies, J Clin Invest 
1995;95:1528-38. 

Roth RA, Kovacina KS, Seta KA, Reinhardt RR, Bondy CA. The insulin receptor-related receptor. In: Draznin, 
B, LeRoith D, eds. The Molecular Biology of Diabetes. Totowa, Humana Press, pp.79-93. 

Schoen TJ, Waldbillig R, Bondy CA, Zhou J, Dhawan R, Mazuuk K, Arnold D, Rodriguez I, Beebe D, Chader 
G. Differential expression and in situ localization of insulin like growth factor binding protein-2 in developing 
chick ocular tissues. Invest Vision Res, in press. 



vo 



HD-00628-06 DEB 



Wang E, Wang J, Chin E, Zhou J, Bondy CA. Cellular patterns of IGF system gene expression in murine 
chondro- and osteogenesis, Endocrinology 1995;136:2741-52. 

Yao DL, West N, Bondy, CA, Brenner M, Hudson L, Zhou J, Collins GH, Webster HdF. Cryogenic spinal cord 
injury induces astrocytic IGF-I expression during myelin regeneration, J Neurosci Res 1995;40:647-59. 

Zhou J, Guidice L, Bondy CA. Regulation and cellular localization of insulin-like growth factor system gene 
expression in the cycling human endometrium, JCEM 1994;79:1723-34. 

Zhou J, ReFeuzo J, Bondy CA. Granulosa cell DNA synthesis is strictly correlated with the presence of IGF-I 
and absence of AP-1 expression in vivo, Molec Endocrinol 1995;9:924-32. 



?i. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00631-06 DEB 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT 180 characters or less. Title must fit on one line between the borders.) 

Neuroendocrine Control of the Stress Response 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator! (Name, title, laboratory, and institute affiliation) 

P.I.: G. Aguilera Head SEP, DEB, NICHD 

Others: A. Kiss Visiting Scientist SEP, DEB, NICHD 

X. Luo NRSA Fellow SEP, DEB, NICHD 

C. Rabadan-Diehl IRTA Fellow SEP, DEB, NICHD 

E. Mancilla Visiting Fellow SEP, DEB, NICHD 



COOPERATING UNITS (if any) 

S. Lolait, LCB, NIMH, NIH 



LAB/BRANCH 



Developmental Endocrinology Branch 



SECTION 

Section on Endocrine Physiology 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 
3.8 



PROFESSIONAL: 

3.8 



OTHER: 





CHECK APPROPRIATE BOXIES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues H (c) Neitlier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project has focused on the mechanism of regulation of hypothalamic and pituitary function during stress . Increases 
in vasopressin (VP) expression in parvicelluUar neurons of the PVN, as well as pituitary VP (Vlb) receptor 
upregulation are important determinants of the increased ACTH responsiveness observed in several chronic stress 
paradigms. Further evidence for the importance of VP was provided by studies during adjuvant induced arthritis in rats, 
showing elevation in VP mRNA in the parvicellular PVN and increases in pituitary VP receptors accompaning marked 
activation of the hypothalamic-pituitary-adrenal (HPA) axis. 

Studies on the regulation of pituitary VP receptors were extended to investigate the mechanism of regulation of Vlb 
receptor expression . Adrenalectomy causes parallel decreases in VP binding and Vlb receptor mRNA, an effect which 
was prevented by glucocorticoid replacement. Similar changes following adrenalectomy were found in Brattleboro VP 
deficient rats indicating that the decreased Vlb receptor expression was not due to adrenalectomy-induced increases in 
parvicellular VP. Increasing pituitary exposure VP by surgical shunting of magnocellular VP to the hypophyseal portal 
circulation caused pituitary VP receptor downregulation without changes in Vlb receptor mRNA levels. Dexamethasone 
administration in intact rats was without effect on Vlb receptor mRNA levels. These data suggest that Vlb receptor 
mRNA levels are directly regulated by glucocorticoids or by hypothalamic factors other than VP. 

Studies to elucidate the mechanism by which stress induces CRH receptors in the hypothalamic paraventricular nucleus 
(PVN) focused on the role of glucocorticoids and neural pathways. While adrenalectomy transiently induced CRH 
receptors in die parvicellular PVN, glucocorticoid administration or withdrawal had no effect on stress induced receptor 
expression. Surgical hemisections of ascending neural pathways induced CRH receptor mRNA in lower brainstem/spinal 
cord projecting neurons of the PVN, but had no effect on basal or stress-induced CRH receptor mRNA in 
hypophyseotrophic parvicellular subdivision. These data show that while glucocorticoids and neural imputs from the 
brain stem and spinal cord influence CRH receptor expression in the PVN, other factors and neural pathways are 
responsible for stress-induced CRH receptor expression. 



PHS 6040 (Rev. 5/92) 



( ■ 



ZOl HD 00631-06 DEB 

Project Description: 

Objectives: 

The aim of this project is to characterize the neuroendocrine components of the adaptation to stress, with 
special emphasis in the mechanism of regulation of the hypothalamic pituitary-adrenal axis. This project involves 
characterization of receptors for the regulatory hormones, elucidation of the mechanism that regulate expression, 
synthesis and secretion of hypothalamic regulatory factors, elucidation of the molecular mechanisms that follow 
hormone receptor interaction, analysis of hormonal secretory patterns and CRT receptor regulation during 
development and manipulations of the hypothalamic-pituitary-adrenal axis. It is expected that this work will 
provide information on the physiological regulation of ACTH secretion and provide a basis for the understanding 
of pathological alterations of the hypothalamic-pituitary-adrenal axis. 

Methods employed: 

The experiments are performed mainly in rats, and include both in vivo and in vitro procedures. In vivo 
experiments include application of various stress paradigms, prolonged infusions of CRH, VP and other regulators 
with osmotic minipumps, blood collection in conscious rats implanted with chronic intravascular catheters, 
implantation of icv cannulae and lesions of specific brain areas using stereotaxic procedures. In vitro procedures 
include measurement of CRH, VP and glucocorticoid receptors by conventional receptor assays and 
autoradiography, detection of neuropeptides by RIA and immunohistochemical techniques, preparation of enzyme- 
dispersed pituitary and brain cells, measurement of hormone release and intracellular messengers, measurement 
of adenylate cyclase and protein kinases, measurement of mRNA for several peptides, receptors and enzymes by 
northern blot, hybridization in solution and in situ, and molecular cloning techniques. 

Progress Report: 

Previous studies of this laboratory demonstrated three types of responses of the hypothalamic-pituitary- 
adrenal axis during adaptation to chronic stress: a) desensitization of the ACTH response to the primary stress, 
but hyperresponsiveness to a novel stim.ulus, b) sustained ACTH response to the primary stimulus and 
hyperresponsiveness to a novel stimulus, and c) minor ACTH responses to the primary stimulus and reduced 
sensitivity to a novel stress. Chronic stress paradigms associated with pituitary hyperresponsiveness showed 
activation of parvicellular CRH and CRHA'^P neurons of the PVN, and increased VP/CRH secretion ratios. The 
activity of magnocellular neurons which provide VP to the neurohypophysis and peripheral circulation is 
unaffected by these stress paradigms. 

The role of hypothalamic CRH and VP in the regulation of the HPA axis was further studied during 
development of adjuvant induced arthritis in the rat, an experimental model associated with activation of the HPA 
axis. irVP in pituitary portal blood increased immediately preceding the onset of arthritis and increases 
progressively peaking at day 16. The increase in VP is associated with a significant increase in the expression 
of VP but not CRH mRNA in the medial parvicellular division of the PVN of arthritic rats. In the presence of 
maximal inflamation there was a decrease in CRH receptor content in the pituitary, whereas VP receptor 
concentration was significantly increased. Basal and CRH plus VP-stimulated ACTH secretion in vitro was higher 
in arthritic rats. The results are consistent with the view that activation of the parvicellular vasopressinergic 
system has an important role in the adaptation of the HPA axis to experimentally-induced chronic stress or 
arthritis. 

Shunting of magnocellular VP and oxytocin (OT) to the pituitary portal circulation by surgical pituitary 
stalk compression (PSC) was used as an experimental model to study the effects of incresing corticotroph 
exposure to VP. The consequences of PSC on hypothalamic and pituitary function were studied by in situ 
hybridization analysis of hypothalamic VP, OT and CRH mRNA levels and pituitary POMC, CRH receptor and 
VP Vlb receptor mRNA levels in rats, with or without ddAVP infusion. 7 days after PSC, there was a marked 



7 



u> 



ZOl HD 00631-06 DEB 

decrease in VP mRNA (83%) and a moderate decrease in OT mRNA (38%) in the hypothalamic supraoptic 
(SON) and paraventricular (PVN) nuclei, probably due to retrograde degeneration of magnocellular neurons. In 
addition, CRH mRNA levels in the parvicellular PVN were decreased by 55% in PSC rats. Correction of diabetes 
insipidus and osmotic alterations by minipump infusion of ddAVP, 5 ng/hr, s.c, caused a further decrease in 
hypothalamic levels of VP and OT mRNA (100% and 60%, respectively), but prevented the decrease in CRH 
mRNA in the PVN. On the other hand, central minipump infusion of VP, up to 3 ng/hr, i.c.v., for 7 days, failed 
to decrease CRH mRNA levels in the PVN. In the pituitary, POMC mRNA levels were unchanged in the anterior 
lobe, but were markedly increased in the intermediate lobe, consistent with dopaminergic denervation of the 
posterior pituitary. In contrast to the decrease in receptor binding observed in PSC rats, mRNA levels for CRH 
and Vlb receptors were similar to control rats, suggesting that receptor downregulation is due to post-translational 
events. The data show that reduced hypothalamic CRH expression contributes to inhibition of the HPA axis 
associated with PSC. The decreases in CRH mRNA levels are not due to increased magnocellular activity or 
elevations in central VP levels, but may involve afferent inputs to the PVN in response to osmotic stimulation, 
or feedback mechanisms secondary to pituitary overstimulation by VP. 

Previous studies of this laboratory have shown that during chronic stress, pituitary vasopressin receptor 
(Vlb) binding and Vlb receptor mRNA undergo changes parallel to the changes in responsiveness of the 
hypothalamic-pituitary-adrenal axis. Further studies were conducted to investigate the mechanisms of regulation 
of pituitary Vlb receptors, the changes in Vlb receptor mRNA levels following adrenalectomy and glucocorticoid 
administration in the rat using Northern blots. Hybridization of pituitary poly[A]RNA was performed using a 363 
random primed ^^P-labeled probe corresponding to the coding region of the rat Vlb receptor cDNA, known to 
reveal two mRNA populations with molecular sizes of 3.7 and 3.2 kb. Consistent with the decreases in pituitary 
VP binding, adrenalectomy caused sustained decreases in both Vlb receptor mRNA populations. After 18 hr 
adrenalectomy, pituitary Vlb receptor mRNA levels were reduced by 77% (3.7 kb band) and 62% (3.2 kb band), 
and after 7 days remained decreased by 65% (3.7 kb band) and 46% (3.2 kb band). Glucocorticoid replacement 
by injection of dexamethasone, 100 ng, s.c. daily, prevented the inhibitory effect of adrenalectomy. 
Dexamethasone administration in intact rats resulted in a significant 37% increase in the 3.7 kb Vlb receptor 
mRNA only after 7 day injections. In Brattleboro rats (di/di), which lack hypothalamic VP, pituitary Vlb receptor 
mRNA levels were also decreased after 7 days adrenalectomy, with reductions of 75%) and 62% for the 3.7 kb 
and 3.2 kb bands, respectively, and this effect was prevented by dexamethasone injection. The parallel changes 
in VP binding and Vlb receptor mRNA following adrenalectomy suggesting that regulation of mRNA levels is 
an important site of control of pituitary VP receptor levels. Although increased VP following adrenalectomy may 
contribute to the reduced Vlb mRNA levels, the results in the Brattleboro rat indicate that Vlb mRNA levels are 
directly regulated by glucocorticoids or by hypothalamic factors other than VP. 

Work of this laboratory has shown a stress-specific induction of hypothalamic CRH receptors, with 
increases in the parvicellular PVN during physical-psychological stress, and in the magnocellular PVN and SON 
after osmotic stimulation. These studies were extended to study the effect of glucocorticoids and afferent neural 
pathways on the regulation of CRH receptor expression in the PVN. 

In situ hybridization studies showed that adrenalectomy transiently increased CRH receptor mRNA expression 
in the PVN, from near undetectable levels in controls to 56.4 ± 4.5 (fransmittance values) after 18 hr 
adrenalectomy, an effect which was prevented by glucocorticoid replacement. Longer term adrenalectomy had 
no effect on CRH receptor mRNA levels in the PVN (2.111.5 and 2.0+0.7, after 4 and 6 days, respectively). In 
intact rats, 4 hr after immobilization for one hr or a single i.p. hypertonic saline injection, CRH receptor mRNA 
in the PVN markedly increased (p<0.01), an effect which was unchanged by adrenalectomy (4 or 6 days) or by 
dexamethasone injection, 100 |ag at -14 and 50 (ig at -1 hr, prior to stress. The data show that while stress- 
stimulation of CRH mRNA in the PVN is glucocorticoid-independent, basal levels are likely to be under dual, 
transcriptional and post-transcriptional control by glucocorticoids. 

The role of afferent innervation on CRH mRNA and CRH receptor mRNA levels in the PVN was studied 
in rats under control and stress conditions. Groups of rats were subjected to unilateral transections of the stria 
terminalis (ST), the medial forebrain bundle at the rostral hypothalamic level (RMFB), or the lower brainstem 



?^ 



ZOl HD 00631-06 DEB 

through the medulla oblongata between the obex and the locus coeruleus (MH). Twelve days after surgery, each 
group of rats was further divided into controls (basal conditions) and stressed (1 hr immobilization), before 
collecting brains for mRNA analysis by in situ hybridization histochemistry. While ST and RMFB cuts had no 
effect on basal CRH mRNA levels in the PVN, MH decreased CRH mRNA in the PVN ipsilaterally to the knife 
cut but it was without effect on the contralateral side (-40% and -37% vs contralateral and sham operated, 
respectively, p< 0.01). Acute stress (rats were killed 3 hr after immobilization) increased CRH mRNA levels by 
about 30% bilaterally, an effect which was unchanged by ST, RMBF or MH. Under basal conditions CRH 
receptor mRNA levels were undistinguishable from the surrounding background in the PVN of sham operated 
controls, ST and RMFB operated rats. However, brain stem hemisection resulted in clear expression of CRH 
receptor mRNA in the dorsal, medial-ventral and lateral parvicellular subdivisions of the PVN, ipsilateral to the 
transection. CRH neurons in these subdivisions project to the lower brain stem and the spinal cord. Expression 
of CRH receptor mRNA in the medial-dorsal and anterior parvicellular divisions (CRH neurons with median 
eminence projections) was not affected by this MH cut. In these subdivisions, immobilization stress markedly 
increased CRH receptor mRNA levels but it did not influence MH cut-induced CRH receptor expression. ST and 
RMFB were without effect on PVN CRH receptor mRNA levels under basal or stress conditions. Oxytocin (OT) 
and vasopressin (VP) mRNA levels in the magnocellular subdivision of the PVN were unchanged after 
immobilization, or following ST, RMFB or MH cuts, whereas OT mRNA in the medial-ventral and caudal 
parvicellular subdivisions was decreased by 52% after MH cut. The data demonstrate that 1) basal CRH mRNA 
levels in the PVN are under tonic stimulatory influence of the lower brain stem (and/or spinal cord) afferents, 2) 
CRH receptor mRNA expression in PVN subdivisions (pituitary vs lower brainstem/spinal cord projecting 
neurons) is under different control mechanisms, and 3) immobilization stress induced changes in CRH mRNA 
and CRH receptor mRNA levels are mediated either by neural inputs from brain areas other than those 
investigated here, or by humoral factors. 

The regulation of CRH receptor expression in the pituitary was ftirther investigated by studying the 
effects of adrenalectomy and glucocorticoids on CRH binding and CRH receptor mRNA levels. CRH receptor 
mRNA levels decreased transiently following adrenalectomy (-62%) after 18 hr), returning to basal levels after 
4 or 6 day adrenalectomy, whereas binding of '"l-Tyr-oCRH showed a sustained decrease. The early decrease 
was prevented by glucocorticoid replacement. Similar transient decreases in CRH receptor mRNA were observed 
by Northern blot analysis. In intact rats, dexamethasone (100 |ig s.c.) caused a significant decrease in pituitary 
CRH receptor mRNA levels 2 to 10 hr after injection, returning to basal levels after 15 hr. On the other hand, 
dexamethasone (5 to 300 ng, s.c.) had no effect on pituitary CRH receptor mRNA levels 18 hr after injection. 
In the pituitary, changes in hypothalamic corticotropin releasing factors probably play a major role controlling 
CRH receptor mRNA levels during manipulations of circulating glucorticoids levels. In addition, the inability of 
long term adrenalectomy and glucocorticoid administration to modify pituitary CRH receptor mRNA levels 
suggests that CRH receptor downregulation observed under these experimental conditions depends mainly on 
franslational and post-translational events rather than receptor mRNA levels. 

Significance to Biomedical Research and the Program of the Institute: 

Exposure to different degrees of sfressfiil stimuli is a prevalent problem in society. Stressfiil stimuli are 
integrated in the central nervous system and lead to a number of responses that include behavioral, visceral and 
hormonal adaptation. The changes in the hypothalamic-pituitary axis during prolonged stress can result in 
alteration of the secretion of most pituitary hormones with consequent alterations in metabolic, immune and 
reproductive fianction. Elucidation of the physiological mechanisms involved in the stress response will be 
important for the prevention and management of clinical conditions that involve disorders of neuroendocrine 
regulation. 

Proposed Course: 

Studies on the regulation of pituitary CRH and VP receptor expression will be extended to the molecular 
mechanisms. Initial studies will determine the second messengers involved in the regulation of the mRNAs of 






ZOl HD 00631-06 DEB 

these receptors in primary cultures of pituitary cells and in the cell line ATt-20. The cloning of the promoter 
region of the CRH receptor and VP receptor genes to study the transcriptional regulation of these receptors is a 
priority. A single genomic clone containing about lOkb upstream of the 5 'untranslated region of the VP receptor 
has been obtained and is in the process of being sequenced. Studies are also in progress to determine the 
mechanisms involved in the dramatic changes in CRH receptor expression in the PVN. This include the role of 
glucocorticoids and afferent innervation of the PVN. The physiological role of CRH in regulating PVN function 
during stress is under study using CRH antagonists and antisense oligonucleotides to block the expression of the 
receptor. 

Studies on the role of glucocorticoid feedback will be extended to the differential analysis of changes 
in glucocorticoid and mineralocorticoid receptors in pituitary and brain, as well as changes in 1 113-dehydrogenase, 
the enzyme which determines the interaction of glucocorticoids with the receptors. The expression of transcription 
factors which regulate the interaction of glucocorticoid receptors with glucocorticoid regulatory elements in the 
POMC gene will be studied in pituitary cells isolated from control and stressed rats and in cell lines. Studies will 
be continued to determine the role of catecholamines and neuropeptides in the expression and release of CRH and 
VP in the hypothalamus. 

Publications 

Aguilera G. Regulation of ACTH secretion during stress, Frontiers in Neuroendocrinology 1994;15:321-50. 

Aguilera G, Rabadan-Diehl C, Luo X, Kiss A. Regulation of pituiatry ACTH secretion during stress: role of 
corticotropin releasing hormone and vasopressin receptors. In: McCarty R, Aguilera G, Sabban E, Kvetnansky 
R, eds. Stress: molecular genetic and neurobiological advances. New York, Gordon and Breach, in press. 

Chowdry HS, Larsen PJ, Harbuz MS, Jessop DS, Aguilera G, Eckland DJA, Lightman SL. Evidence for arginine 
vasopressin as the primary activator of the HPA axis during adjuvant-induced arthritis, Br J Pharmacol, in press. 

Kapcala L, Aguilera G. Modulation of corticotropin-releasing hormone stimulated cyclic adenosine 
monophosphate production by brain cells. Brain Res 1995;678:207-12. 

Kiss A, Jezova D, Aguilera G. Activity of the hypothalamic pituitary adrenal axis and sympathoadrenal system 
during food and water deprivation in the rat. Brain Res 1994;663:84-92. 

Luo X, Kiss A, Rabadan-Diehl C, Aguilera G. Regulation of hypothalamic and pituitary corticotropin releasing 
hormone receptor mRNA by adrenalectomy and glucocorticoids. Endocrinology, in press. 



76 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl HD-00632-06 DEB 



PERIOD COVERED 



October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.! 

Physiological Actions of the Renin-Angiotensin-Systetn 



PRINCIPAL INVESTIGATOR fUst other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: G. Aguilera Head SEP, DEB, NICHD 

Others: A. Kiss Visiting Scientist SEP, DEB, NICHD 

X. Luo NRSA Fellow SEP, DEB, NICHD 



COOPERATING UNITS lif any) 

None 



LAB/BRANCH 



Developmental Endocrinology Branch 



SECTION 



Section Endocrine Physiology 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, MP 20892 



TOTAL STAFF YEARS: 
0.7 



PROFESSIONAL: 

0.7 



OTHER: 





CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects D (b) Human tissues E (c) Neither 
D (a1) Minors 
D (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The purpose of this project is to study physiological and pathological aspects of the renin-angiotensin system, 
emphasising its role in circulatory homeostasis and development. During the past year studies have focused on a) the 
role of type- 1 angiotensin II (Ang II) receptors located in parvicellular corticotropin-releasing hormone CRH and 
CRHA^P cells in the hypothalamic paraventricular nucleus (PVN), and b) the consequenses of chronic stress on the 
peripheral renin-Ang-aldosterone system. 

a) The regulation of Ang II receptors in the PVN by glucocorticoids and the role of Ang II regulating the 
responsiveness of the HPA axis during stress was studied by correlating Ang II receptor expression in the PVN with 
changes in activity of the HPA axis. Various stress paradigms increased Ang II receptor expression in the 
parvicellular subdivision of the PVN in similar fashion irrespective of their effect on the activity of the HPA axis. 
Ang II receptor expresion in parvicellular cells was reduced by adrenalectomy and increased by glucocorticoid 
administration. The stimulatory effect of stress on Ang II receptor expression in the PVN was prevented by 
adrenalectomy, indicating that glucocorticoids are necessary for the effect of stress. These data support a role for Ang 
II in the central mechanisms of adaptation to stress, but indicate that Ang II does not play a role controlling the 
responsiveness of the HPA axis. 

b) Repeated physical-psychological stress reduced basal and stimulated aldosterone secretion in spite of elevated 
basal plasma renin activity. The increases in plasma renin activity were associated with increases in renin mRNA 
expression in the kidney due to repeated sympathetic stimulation. In vitro studies revealed that while the early 
aldosterone biosynthetic pathway is increased, Ang II receptor content, mRNA levels and activity of aldosterone 
synthetase are decreased in chronically stress rats. The data provides evidence for a role of chronic stress in the 
development of hyperreninemic hypoaldosteronism. 



7V 



PHS 6040 (Rev. 5/92) 



ZOl HD-00632-06 DEB 

Project Description: 

Objectives: 

The purpose of this project is to study the role of the renin-angiotensin-system in mammalian physiology 
with special emphasis on its role in the circulatory homeostasis and growth and development. Studies include 
characterization and mapping of the distribution of Ang II receptors subtypes and their function in the nervous 
system, endocrine organs and other peripheral tissues, and elucidation of the molecular mechanisms involved in 
the regulation of cellular function by the peptide. Studies on the control of mineralocorticoid secretion include 
analysis of the interaction of Ang II with other regulators, at the systemic and cellular level and to define the 
mechanisms responsible for changes in adrenal sensitivity to the peptide during changes in sodium intake. 

Methods employed: 

The majority of experiments are performed in the rat, and involve both in vivo and in vitro procedures. 
In vivo procedures include modifications of dietary electrolytes, administration of Ang II and inhibitors of the 
renin-AII system and blood sampling in conscious rats with chronically implanted intravascular catheters. In vitro 
procedures include preparation of enzyme-dispersed fetal fibroblasts and adrenal glomerulosa cells, measurement 
of Ang II receptors in dispersed cells, membrane rich fractions and slide mounted tissue sections by conventional 
assays and by autoradiography; measurement of steroid production and intracellular messengers in isolated cells 
under basal and stimulated conditions; analysis of cellular proteins by gel electrophoresis and immunoblot 
techniques, and determination of mRNA by Northern blot, hybridization in solution and in situ hybridization. 

Major findings: 

Angiotensin II is a major regulator of adrenal mineralocorticoid secretion and blood pressure. In addition, 
components of the renin-angiotensin system are present in the brain and a number of peripheral tissues where it 
acts as a neurotransmitter or as a paracrine regulator. The effects of Ang II are mediated by plasma membrane 
receptors located in the adrenal zona glomerulosa and other target tissues. Two major subtypes of Ang II receptors 
have been identified, ATI, which are coupled to calcium-phospholipid dependent signalling systems, and AT2, 
with yet undefined actions. Previous studies of this laboratory have described the topographic localization and 
characterization of Ang II receptor subtypes in the brain and peripheral tissues. 

Studies of this group using double staining in situ hybridization techniques have shown that Ang I type 
1 receptors (ATI) in the hypothalamic paraventricular nucleus (PVN) are located primarily in corticotropin 
releasing hormone (CRH) neurons of the parvicellular subdivision. Further studies were conducted to investigate 
the role of Ang II regulating the hypothalamic-pituitary adrenal (HPA) axis, by correlating AT, receptor 
expression levels in the PVN with the known changes in activity of the HPA axis under different stress 
paradigms, and manipulation of circulating glucocorticoids. AT, receptor mRNA was measured by in situ 
hybridization using ''S-labelled cRNA probes and Ang II binding by autoradiography using '^'l[Sar',Ile^]AII in 
slide mounted hypothalamic sections. AT, receptor mRNA levels and Ang II binding in the PVN were reduced 
by about 20% 18 hr after adrenalectomy remaining at these levels up to 6 days thereafter. This effect was 
prevented by corticosterone administration in the drinking water, or dexamethasone injection (100 |ig, s.c, daily). 
Conversely, dexamethasone injection in intact rats caused a 20% increase in AT, receptor mRNA in the PVN. 
AT, receptor mRNA and binding in the P^/N increased 4 hr after exposure to stress paradigms associated with 
activation of the HPA axis (immobilization for one hr, or i.p. injection of 1.5 M NaCl), and remained elevated 
after repeated daily stress for 14 days. Unexpectedly, two osmotic stress models associated with inhibition of the 
HPA axis (60 hr water deprivation or 12 days of 2% saline intake) also resulted in increased AT, receptor mRNA 
levels and Ang II binding in the parvicellular PVN. In intact rats, the stimulatory effect of acute stress on AT, 
receptor mRNA in the PVN was significantly enhanced by dexamethasone administration (100 |ig, s.c, 14 hr and 
1 hr prior to stress), while in adrenalectomized rats, with or without glucocorticoid replacement, stress reduced 
rather than increased, AT, receptor mRNA. Dexamethasone, 100 |ig, injected sc within 1 min the beginning of 



'?i 



ij 



ZOl HD-00632-06 DEB 

immobilization in adrenalectomized rats, increased AT, receptor mRNA in the PVN to levels significantly higher 
than those after dexamethasone alone, indicating that the stress induced glucocorticoid surge is required for the 
stimulatory effect of stress on AT, receptor mRNA. The data suggests that AT, receptor expression in the PVN 
is under dual control during stress: stress-activated inhibitory pathways and the stimulatory effect of 
glucocorticoids. The lack of specificity of the changes in AT, receptor expression in the PVN following stressors 
with opposite effects on ACTH secretion (osmotic and physical-psychological stress) does not support a role for 
Ang II as a major determinant of the response of the HPA axis during stress. 

Previous studies have shown that Ang II is the major regulator of aldosterone secretion. ACTH is also 
a potent stimulant in acute conditions but its prolonged administration markedly inhibits aldosterone secretion. 
Circulating levels of both Ang II and ACTH and therefore aldosterone increase in response to stress. Studies were 
conducted to determine the consequenses of chronic stress on the renin-angiotensin-aldosterone system by analysis 
of plasma hormone levels, kidney renin mRNA levels, and adrenal Ang II receptors and steroidogenesis in rats 
under repeated immobilization (2 hr daily), or ip injection of 1.5 M NaCl (ipHS) for 14 days. 24 hr after the last 
stress in both stress models, plasma aldosterone levels were reduced in spite of significant increases in PRA. 
Repeatedly ipHS rats showed PRA responses to acute immobilization similar to controls, but markedly reduced 
plasma aldosterone responses. Concomitant with the increases in PRA, renin mRNA levels in the kidney were 
significantly increased in ipHS rats, and these increases were prevented by fi-adrenergic receptor blockade with 
propranolol. In isolated adrenal glomerulosa cells from chronically stressed rats, maximum aldosterone responses 
to All, ACTH and 8-Br-cAMP were significantly decreased, whereas pregnenolone responses were increased. 
P450-aldosterone synthetase mRNA levels and binding of '^'l[Sar',Ile*]AII were significantly reduced in the 
adrenal zona glomerulosa of stressed rats. These studies show that chronic repeated stress leads to renin 
stimulation due to sympathetic activation, and inhibition of aldosterone secretion due to inhibition of the late 
steroidogenic pathway. The data provides evidence for a role of chronic stress in the development of 
hyperreninemic hypoaldosteronism. 

Significance to Biomedical Research and the Program of the Institute 

The prominence of Ang II receptor expression during crucial periods of fetal and postnatal growth open 
new perspectives about the importance of the renin-AII system during development. Elucidation of the function 
of these novel receptors during development is of critical importance, especially in view of the increasing clinical 
use of drugs that affect the ftinction of the renin-AII system. The studies on the central actions of Ang II are 
likely to uncover novel regulatory mechanisms in neuroendocrine function, including modulatory actions of the 
activity of the HPA axis, growth and reproductive function. In addition elucidation of the mechanism of regulation 
of adrenal glomerulosa cell ftinction is relevant to understanding the pathogenesis and treatment of disorders 
involving alterations of mineralocorticoid secretion. 

Proposed Course: 

Future emphasis of this project will be placed in the elucidation of the neuroendocrine ftinctions of All. 
Based on the topographic localization of Ang II receptor mRNA in the PVN, the hypothesis to be tested is that 
Ang II has a modulatory effect on the function of the CRH neuron, and possibly on somatostatin and 
dopaminergic neurons of the PVN. Further in situ hybridization histochemistry studies will be performed to 
determine whether Ang II receptors are expressed in somatostatin and dopaminergic neurons in the PVN. The 
function of Ang II in the regulation of the HPA axis, growth hormone and prolactin secretion will be studied 
using Ang II receptor blockade with specific Ang II antagonists and antisense oligonucleotides during 
neuroendocrine manipulations, and by analysis of changes in Ang II receptor expression in relevant brain areas 
during manipulations of the HPA axis or gonadal hormones in male and female rats. 



7U 



ZOl HD-00632-06 DEB 

Publications 

Aguilera, G, Kapur, S, Feuillan P, Akbasak, B and Bathia A. Developmental changes in angiotensin II receptor 
subtypes and AT, receptor mRNA in rat kidney, Kidney Int 1994;46:973-9. 

Aguilera G and Kiss A. Angiotensin II and the regulation of the hypothalamic-pituitary-adrenal axis and 
vasopressin secretion. In: Raizada M, Phillips MI, Sumners C, eds. Recent advances in anngiotensin II receptors. 
New York, Plenum Publishing Corp, in press. 

Aguilera G, Kiss A, Luo X. Increased expression of type-1 angiotensin II receptors in the hypothalamic 
paraventricular nucleus following stress and glucocorticoid administration; J Neuroendocrinol, in press 

Aguilera G, Kiss A, Luo X and Sunar-Akbasak B. The renin-angiotensin system and the stress response, Ann NY 
Acad Sci, in press 

Aguilera G, Kiss A and Sunar-Akbasak B. Hyperrreninemic hypoaldosteronism following chronic stress in the 
rat, J Clin Invest, in press 

Aguilera G, Young WS, Kiss A, and Bathia A. Direct regulation of hypothalamic corticotropin releasing hormone 
neurons by angiotensin II, Neuroendocrinology 1995;61:437-44. 

Burton JY, Aguilera G, Gross K, Sigmund CD. Differential expression of type lA and type 1-B angiotensin II 
receptor mRNA in the mouse, Am J Physiol 1994;267:E260-7. 



cU 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00633-05 DEB 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 



Ovarian folliculogenesis 



PRINCIPAL INVESTIGATOR {List other professional personnel below the Principal Investigator.) IName, title, laboratory, and institute affiliation) 

P.I. L.M. Nelson Senior Clinical Investigator SGR, DEB, NICHD 



Others: 



Z.B. Tong 
J. Anasti 
S. Nair 
R. Maity 



Visiting Fellow 
Clinical Associate 
NRSA Fellow 
Visiting Fellow 



SGR, DEB, NICHD 
SGR, DEB, NICHD 
SGR, DEB, NICHD 
SGR, DEB, NICHD 



COOPERATING UNITS (if any) 

J. Dean, Mammalian Developmental Biology Section, Laboratory of Cellular and Developmental Biology, NIDDK; 
R. Caspi, Laboratory of Immunology NEI 



LAB/BRANCH 

Developmental Endocrinology Branch 



SECTION 



Gynecologic Research Section 



INSTITUTE AND LOCATION 



National Institute of Child Health and Human Development 



TOTAL STAFF YEARS: 

3.0 



PROFESSIONAL: 

3.0 



OTHER: 





CHECK APPROPRIATE BOX(ES) 

(a) Human subjects 
H (a1) Minors 
D (a2) Interviews 



£] (b) Human tissues D (c) Neither 



SUMMARY OF Vi/ORK (Use standard unreduced type. Do not exceed the space provided.) 

This project seeks to improve the clinical care available to patients with disorders of ovarian follicle fiinction and 
ovulation through research using animal models and clinical protocols. In pursuing this goal, we expect to expand 
understanding of the ovarian follicle in health and disease. We have focused on premature ovarian failure, a 
condition that prematurely terminates ovarian function and fertility in 1% of women. During this period we found 
that femoral neck bone density is significantly reduced in women with premature ovarian failure. Surprisingly, more 
than one-half of our patients had a bone density low enough to cause a threefold increase their risk of fracture. 
Strikingly, we found luteinized graafian follicles in all the antral follicles we biopsied in 6 patients with 
karyotypically normal spontaneous premature ovarian failure. Therefore, luteinized graafian follicles account for at 
least 60% of the antral structures imaged (95% confidence limit). Inappropriate luteinization of graafian follicles 
appears to be a major pathophysiologic mechanism in patients with karyotypically normal spontaneous premature 
ovarian failure. We have particular interest in autoimmune ovarian failure. Using an animal model, we found that 
autoantibodies from mice with experimental autoimmune oophoritis bind to a 120 kd protein that is specific to the 
oocyte cytoplasm. By screening a mouse ovarian cDNA expression library with this serum, we identified a novel 
oocyte-specific gene that may represent the inciting antigen in this disease. Also, we showed that the inciting 
ovarian antigen in murine experimental autoimmune oophoritis appears to be produced independently of gonadotropin 
stimulation. By administering gonadotropin stimulation or suppression to neonatally thymectomized mice we did not 
alter the course of the disease. During this period we also found that the lymphocytic infiltration in murine 
experimental autoimmune oophoritis is primarily localized in the stroma and theca, and does not involve a direct 
lymphocytic attack against intact oocytes. Intriquingly, our investigations in murine post-thymectomy polyglandular 
autoimmunity suggest that neonatal thymectomy causes a deficiency of CD4+ T helper 1 (Thl) activity that persists 
into adulthood. In ongoing work we are testing the hypothesis that this Thl-deficient state induces the autoimmune 
disease. We will also determine, using transgenic technology, if the novel oocyte-specific gene is the inciting ovarian 
antigen in murine experimental autoimmune oophoritis. Future clinical directions involve developing strategies to 
increase bone density in women with premature ovarian failure, a treatment to prevent inappropriate luteinization in 
patients with premature ovarian failure, and a protocol to test the hypothesis that inappropriate luteinization plays a 
role in the infertility of maturing women. 



PHS 6040 (Rev. 5/92) 



ZOl HD 00633-05 DEB 

Project Description 

Objectives: 

This project seeks to improve the clinical care available to patients with disorders of ovarian follicle function. 
We approach this goal from abroad perspective, aiming to advance knowledge of ovarian follicle physiology and 
pathophysiology through research using animal models of human reproductive disorders, and through clinical 
protocols evaluating diagnostic and therapeutic methods for these patients. The consolidating goal here is to 
develop methods to restore ovulation in patients currently unresponsive to available therapies. In the process of 
pursuing these goals, we expect to expand essential understanding of the ovarian follicle in health and disease. 
We now have specific interest in patients with premature ovarian failure. Approximately one percent of women 
experience ovarian failure prior to age 40. Although symptoms related to estrogen deficiency in these patients 
can be treated with estrogen replacement therapy, there is no known treatment to restore ovulatory function and 
make these women fertile. This problem has had greater impact in recent years. As modem women more 
commonly delay childbearing, more women will experience infertility as the most significant problem related to 
premature ovarian failure. 

Our specific research objectives during the current period were directed along three main avenues. 

1. Clinical research in premature ovarian failure 

a) Examine bone density in a group of these patients. 

b) Study the function of ovarian antral follicles in these women. 

c) Define the histopathology of these antral follicles to gain insight into the mechanism of ovarian follicle 
dysfunction. 

d) Evaluate treatments for patients with autoimmune oophoritis. 

2. Basic research to develop clinical diagnostic markers for autoimmune premature ovarian failure 

a) Investigate circulating interfering immunoglobulins as a cause of premature ovarian failure. 

b) Explore antibodies against the zona pellucida as a marker for autoimmune premature ovarian failure. 

c) Examine the frequency of antibodies against human recombinant thyroid peroxidase and gastric parietal cells 
m patients with premature ovarian failure. 

3. Basic research in murine post-thymectomy autoimmunitv, an animal model of autoimmune premature ovarian 
failure 

a) Characterize the inciting ovarian antigen in this model. 

b) Define how neonatal thymectomy perturbs the developing immune system so as to induce autoimmunity. 

c) Evaluate the mouse major histocompatibility system (H2) as a determining factor in strain-specific susceptibility 
to post-thymectomy autoimmune oophoritis. 



O^' 



ZOl HD 00633-05 DEB 

Methods Employed: 

Clinical premature ovarian failure 

Premature ovarian failure is a condition causing amenorrhea, hypoestrogenism, and elevated gonadotropins in 
women less than age 40. Patients with known abnormal karyotypes or evidence to suggest iatrogenic ovarian 
failure are not included in our studies. 

Patients with premature ovarian failure are evaluated clinically to include: measurement of sex steroid hormones, 
gonadotropins, free T4, and thyroid stimulating hormone by radioimmunologic methods; karyotype analysis on 
cultured lymphocytes; Human Leucocyte Antigen (HLA) analysis by immunophenotyping using standard 
microcytotoxic typing with locally prepared HLA-DRl-10 antiserum trays; determination of bone density by dual 
energy x-ray absorptiometry; and evaluation of ovarian structure by ultrasonography. Patients may also undergo 
a three hour glucose tolerance test and ACTH stimulation test. Autoimmune activity is determined by 
immunohistochemistry, hemagglutination assay, and enzyme linked immunosorbent assay. Antiovarian antibodies 
in women are detected by indirect immunofluorescence using M. cynomolgus ovary as tissue substrate. These 
clinical investigations involve the participation of either healthy human subjects or patients. 

Previous investigators have used ovarian wedge biopsy in the evaluation of premature ovarian failure. However, 
wedge biopsy was originally developed as a technique used in polycystic ovarian syndrome, a condition associated 
with abnormally large ovaries. Patients with premature ovarian failure have small ovaries, and wedge biopsy in 
this condition has tremendous sampling error. Therefore, rather than performing wedge biopsy at a random time, 
we have perform directed ovarian follicle biopsy when an ovarian antral follicle was visualized by pelvic 
ultrasound. Ovarian biopsy is performed under general anesthesia by minilaparotomy or laparoscopy. Biopsy 
specimens are processed and stained routinely with hematoxylin and eosin, and immunohistochemical studies are 
performed to detect lymphocytic infiltration. 

We test for interfering antibodies in women with premature ovarian failure using a recombinant system expressing 
human FSH and LH receptors. A mouse adrenal cell line transfected with the human FSH receptor gene (Yl- 
hFSHR) exhibits a dose dependent increase in progesterone when exposed to hFSH. An embryonal kidney cell 
line transfected with the human LH receptor gene (hLHR-293) exhibits a dose dependent increase in cAMP when 
exposed to hLH. We isolated immunoglobulins from patients with premature ovarian failure and normal women. 
Anti-gonadotropin polyclonal antibodies isolated in the same manner were used as a positive control. We 
stimulated YI-hFSHR and hLHR-293 cells with hFSH or hLH in the presence of these immunoglobulins and 
determined the resulting progesterone or cAMP response. 

Murine post-thymectomy polyglandular autoimmunity 

Thymectomy performed two to four days after birth induces murine polyglandular autoimmunity in a strain- 
specific maimer. Mice may develop autoimmune thyroiditis, autoimmune gastritis, or autoimmune oophoritis, 
a constellation of autoimmunity that is seen in the clinic. We work primarily with (C57/BL6 x A/J)F1 hybrids 
(B6A) because 90% of B6A mice develop experimental autoimmune oophoritis after neonatal thymectomy. 

To characterize the autoantibodies in murine experimental oophoritis we use immunoblotting. Ovarian proteins 
are prepared for electrophoresis on SDS-PAGE gel, transferred to nitrocellulose membrane, incubated with sera 
from thymectomized or control mice, and developed with secondary antibody using immunoperoxidase. To 
identify the ovarian proteins that bind antibody, we screen a mouse ovarian cDNA library with serum from mice 
with high-titer ovarian autoantibodies. 

Previous cell transfer studies, using cells from spleens of normal adult mice, have shown that mice thymectomized 
as neonates are deficient in a CD4+ T cell population that can prevent autoimmunity. We seek to determine if 
this is a deficiency ui CD4+ T helper 1 (THl) or T helper 2 (TH2) activity. To characterize CD4+ THl and TH2 



8u 



ZOl HD 00633-05 DEB 

activity, we stimulate CD4+ splenic lymphocytes in vitro by engaging CD3 and CD28 with specific antibodies. 
We measure interferon-gamma (INF-gamma) production, a measure of CD4+ THl cell activity, by enzyme-linked 
immunosorbent assay (ELISA). We measure interleukin-4 (IL-4) production, a measure of TH2 cell activity, 
by utilizing a proliferation assay employing an IL-4-dependent cell line. We enrich for CD4+ T cells by magnetic 
separation and confirm the separation by flow cytometry. To characterize the location of the inciting ovarian 
antigen in this model we stain ovaries with immunoperoxidase immunohistochemistry using monoclonal 
antibodies to CD45, a cell surface protein specific to mouse leukocytes. We evaluate the importance of the mouse 
major histocompatibility system by performing neonatal thymectomy on congenic strains of mice that differ only 
at the H2 locus. 

Major Findings : 

1. Clinical premature ovarian failure 

a. Bone density 

We found that, based on bone density measurements, over one-half of our patients with premature ovarian failure 
have a threefold increased risk of hip fracture. This is despite the fact that 85% of these patients had been taking 
estrogen replacement therapy. We found that 48 of 89 women with premature ovarian failure (54%) had a bone 
density at the femoral neck less than one standard deviation below the mean of normal age-matched women 
(p<0.001, chi square goodness of fit test). A bone density less than one standard deviation below the mean is 
associated with a threefold increase in the risk of fracture. 



b. Antral follicle ftmction 

At one time it was assumed that premature ovarian failure was caused by depletion of ovarian follicles, and, like 
menopause, is an irreversible condition. We continue to collect evidence to refute this view. 

Many patients with karyotypically normal spontaneous premature ovarian failure appear to have a store of 
primordial follicles that are appropriately "timed" to become activated over an extended period, but the 
redundancy in follicle number needed to assure the constant availability of a cohort of developing follicles is 
deficient. Women require the constant availability of a cohort of developing follicles to maintain regular 
ovulatory menstrual cycles. 

We detected ovarian follicle function in approximately 50% of patients with karyotypically normal spontaneous 
premature ovarian failure by measuring serial estradiol blood levels over 4 months. Furthermore, we imaged an 
antral follicle by sonography in over 40% (27/65) of our patients with premature ovarian failure. The median 
ovarian follicle size was 8 mm (range 3 to 46). Interestingly, the probability of detecting a follicle did not appear 
to decline as the time from diagnosis increased. Even patients more than six years since diagnosis had antral 
follicles detected. We know from previous work that follicles are few and far between in ovarian biopsies taken 
from patients with karyotypically normal spontaneous premature ovarian failure. 

The antral follicles in these patients are endocrinologically active, and not mere atretic remnants of follicles. 
Serum estradiol was significantly greater when an antral follicle was present (p<0.01), and this is evidence that 
these structures are indeed endocrinologically active. But the follicles in these patients were not functioning 
normally. We could detect only a weak trend toward a positive correlation between serum estradiol levels and 
follicle diameter in women with karyotypically normal spontaneous premature ovarian failure (r = 0.24, p < 0.10). 
By contrast, in normal women serum estradiol levels have a strong positive correlation with follicle diameter (r 
= 0.91, p < 0.005). During the follicular phase in normal women serum progesterone levels are also strongly 
correlated with follicle diameter (r = 0.83, p < 0.005). We did not detect a correlation between serum 
progesterone levels and follicle diameter in patients with premature ovarian failure. 



tlr' 



ZOl HD 00633-05 DEB 

With this evidence of abnormal follicle function, we biopsied ovarian follicles in a few patients under an approved 
protocol to gain insight into the mechanism causing dysfunction. We sought to determine if autoimmune 
oophoritis was impairing follicle function or was this some other mechanism? 

c. Antral follicle histology 

Strikingly, we found luteinized graafian follicles in all the antral follicles we biopsied in 6 patients with 
karyotypically normal spontaneous premature ovarian failure. Therefore, luteinized graafian follicles account 
for at least 60% of the antral structures imaged (95% confidence limit). Inappropriate luteinization of graafian 
follicles appears to be a major pathophysiologic mechanism in patients with karyotypically normal spontaneous 
premature ovarian failure. 

In these patients, apparently due to an inadequate cohort of follicles, LH fails to be suppressed into the normal 
range, and this leads to inappropriate luteinization of the few graafian follicles capable of responding to FSH 
stimulation. A properly timed LH surge luteinizes a mature follicle, reduces granulosa cell mitogenic activity, 
and induces changes in the steady state level of mRNAs for enzymes involved in progesterone production. 
Inappropriate luteinization of a growing follicle would thus be expected to impair follicle growth and reduce 
estradiol production. The elevation of serum LH during the normal menopausal transition is thought to result 
from inadequate negative feedback due to the reduced number of follicles. In a normal menstrual cycle, the 
cohort of atresia-destined follicles may play a more important role than has been recognized. This cohort 
maintains a proper gonadotropin environment and thus prevents premature luteinization. 

d. Treatment of autoimmune premature ovarian failure 

Two previous studies, one by our laboratory and one by a laboratory in Boston, have demonstrated that patients 
with premature ovarian failure as a group have increased T lymphocyte activation in peripheral blood. This 
suggests autoimmunity plays a role in a substantial portion of these patients. However, we have no serum marker 
to accurately identify individual women who have premature ovarian failure on an autoimmune basis. 

As part of a controlled study evaluating alternate-day prednisone therapy for patients with biopsy-proven 
autoimmune oophoritis, we biopsied developing antral ovarian follicles in six women. Despite our use of 
sensitive immunohistochemical methods, we did not find autoimmune oophoritis in any ovarian follicle biopsies 
performed on these patients. We have, therefore, modified our patient selection criteria to include patients with 
other evidence to suggest autoimmunity. Also, based on our findings in the mouse model, we suspect the disease 
can sometimes be sequestered in the ovarian hilum. Therefore, we have modified our ovarian biopsy technique. 
We are currently evaluating a novel laparoscopic ovarian biopsy strategy that will be certain to obtain a portion 
of the ovarian hilum. 

During this period we completed a prospective, double blind placebo-controlled trial evaluating danazol treatment 
for patients with premature ovarian failure. We tested the hypothesis that a period of ovarian rest might reduce 
antigenic load and allow ovarian function to return after the suppression is withdrawn. Danazol is a weak 
androgen that suppresses gonadotropins and has known favorable immunomodulatory effects. Danazol, did not 
improve ovarian fiinction. 

2. Diagnostic markers for autoimmune ovarian failure 

a. Interfering antibodies 

Circulating immunoglobulins that interfere with the interaction of FSH and LH on their respective receptors 
appear to be an uncommon cause of premature ovarian failure. Despite using human receptors and human 
gonadotropins, we did not detect circulating antibodies inhibiting this interaction in any of 38 patients with 
premature ovarian failure. Therefore, if blocking antibodies that interfere with gonadotropin-receptor interaction 



ao 



ZOl HD 00633-05 DEB 
are a mechanism for premature ovarian failure, they account for a minority of cases (<8%, 95% confidence limit). 

b. Zona pellucida antibodies 

Strikingly, we found that approximately one-third of normal women (n=26) had these antibodies. Thus, the test 
is not helpful to women with premature ovarian failure. However, because one-half of our patients with 
premature ovarian failure had these antibodies, we attempted to improve the specificity of the test. We could not 
improve the test by using higher serum dilutions or simplified outcome measures. 

We are encouraged, however, by that fact that we did not detect zona pellucida antibodies in any of 26 men (p< 
0.001). This is intriguing. This finding supports recent proposals that pre-B cells undergo positive selection 
directed by the presence of surface heavy chain with low affinity to autoantigen. It appears then, that men, 
lacking this self antigen, fail to provide positive selection for these pre-B cells clones. Also, this finding suggests 
that our detection system is specific for zona pellucida, and that further refinement of the assay system by using 
pure human recombinant zona pellucida protein might be useful. 

c. Thyroid peroxidase and gastric parietal cell antibodies 

We found that women with karyotypically normal spontaneous premature ovarian failure have a significantly 
higher incidence of antibodies against human thyroid peroxidase and gastric parietal cells as compared to age- 
matched normal women. The constellation of autoimmunity seen in murine post-thymectomy autoimmune 
oophoritis includes autoimmunity against the thyroid and gastric parietal cells. Therefore, we determined whether 
a similar constellation of autoimmunity was present in patients with premature ovarian failure. Our finding 
suggests this mouse model may indeed have expression in human pathology, and studies to define the inciting 
antigen in this mouse model may have important implications to women with premature ovarian failure. 

Murine post-thymectomy polyglandular autoimmunity 

a. Inciting ovarian antigen 

We have identified a novel oocyte-specific gene. Previously, we found that autoantibodies from mice with 
autoimmune oophoritis bind to a 120 kd protein that is specific to the oocyte cytoplasm. We identified the novel 
gene by screening a mouse ovarian cDNA expression library with this serum. We have demonstrated that the 
message is specific to the ovary by RNAse protection assay, and we have demonstrated that the message is 
specific to the oocyte by in situ hybridization. These mice not only develop high-titer autoantibodies specific to 
oocytes, but ultimately the ovaries become fibrotic and devoid of primordial follicles. These findings implicate 
the oocyte as a primary target of the autoimmune process. 

We have shown that the inciting antigen in murine experimental autoimmune oophoritis appears to be produced 
independently of gonadotropin stimulation of the ovary. These findings are consistent with the hypothesis that 
the inciting antigen is present in oocytes of primary follicles, as suggested by the oocyte specificity of the high- 
titer serum. Primary follicle development does not require gonadotropin stimulation. By administering 
gonadotropin stimulation to neonatally thymectomized mice we did not exacerbate existing disease, or induce an 
earlier onset of severe disease. Furthermore, by administering gonadotropin suppression to neonatally 
thymectomized mice we did not reduce the degree of lymphocytic infiltration or oocyte destruction. 

During this period we found that even the anamnestic development of lymphocytic infiltration in murine 
experimental autoimmune oophoritis was primarily localized in the stroma and theca. On the surface, at least, 
this appears inconsistent with the hypothesis that the inciting antigen is an oocyte protein. In previous work we 
demonstrated that in developing murine autoimmune oophoritis the lymphocytic infiltration was confined to the 
stroma and theca, and not found involving oocytes. Therefore, we investigated the possibility that lymphocytic 
infiltration involving oocytes develops as part of end-stage disease. We transplanted normal syngeneic ovaries 



6B 



ZOl HD 00633-05 DEB 

to B6A mice with confirmed autoimmune ovarian failure, and, as a control, to normal oophorectomized mice. 
Our findings suggest that the ovarian failure in this model is not mediated by a direct lymphocytic attack against 
intact oocytes. Other immune-mediated mechanisms are responsible. The paradoxical development ofhigh-titer 
oocyte-specific antibodies despite the stromal and thecal location of the lymphocytic infiltration remains to be 
explained. 

b. Perturbing the neonatal immune system 

Adoptive transfer of CD4+ splenic lymphocytes from normal adult mice prevents the disease from developing 
in neonatally thymectomized mice. The mechanisms of this regulation, and the reason why the timing of neonatal 
thymectomy is so critical, are unclear. 

We have findings to suggest that 1) neonatal thymectomy induces autoimmunity by impairing development of 
T helper type 1 (Thl) regulation, and 2) the postnatal shift to normal adult Thl/Th2 balance is established by a 
thymus-dependent process. These results hold implications for the pathogenesis, and possibly for the therapy of 
autoimmune polyglandular failure in humans. 

Strikingly, we found that splenic CD4+ cells from adult mice thymectomized as neonates produced an 
inappropriate neonatal-like Th2 predominant response (high levels of IL-4 and low levels of interferon gamma). 
Cells from adult mice sham-operated as neonates produced the expected Thl predominant response seen normally 
in adult mice (low levels of IL-4 and high levels of interferon gamma). 

We also found that administration of interleukin-12, a key cytokine that promotes CD4+ T cell differentiation 
towards the Thl phenotype, restored the adult-like Thl pattern of mice thymectomized as neonates, and 
ameliorated the development of autoimmune oophoritis. We found that postnatal adoptive transfer of adult 
splenocytes to mice thymectomized as neonates had the same effect. 

During the current period we also showed that mice thymectomized as neonates have reduced natural killer (NK) 
cell activity. IL-12 also stimulates NK cells, and increases their production of INF-gamma. This is an additional 
indirect mechanism by which IL-12 promotes CD4+ cell differentiation toward Thl. IL-12, administered after 
neonatal thymectomy, appears to in some way compensate for the lack of thymic function. This raises the 
possibility that the thymus effects the change in the periphery by secretion of IL-12. The message for both the 
p35 and p40 chains of IL-12 are produced in mouse thymus. 

c. Major histocompatibility complex 

During the current period, by using congenic strains of mice in a well-controlled experiment, we showed that 
genetic background plays a more important role than H-2 haplotype in the development of post-thymectomy 
autoimmune oophoritis. The availability of congenic strains of mice makes it possible to separate the effects of 
the genetic background and the specific H-2 major histocompatibility complex. "A" mice (A/J) are highly 
susceptible to post thymectomy autoimmunity, whereas "B" mice (C57 BLIO) are relatively resistant. We showed 
that A.By mice developed severe autoimmune oophoritis equivalent to A/J mice. A.By mice have the "A" genetic 
background, but the "b" H-2 major histocompatibility complex. Therefore, severe disease can develop in "A" 
mice despite the absence of the "a" H-2. Furthermore, we found that BIO.A mice, which have the "B" 
background and the "a" H-2, did not develop disease. Therefore, the presence of the "a" H-2 alone is insufficient 
to convey disease susceptibility. 



^7 



ZOl HD 00633-05 DEB 
Significance to Biomedical Research and the Program of the Institute: 
Clinical premature ovarian failure 

a. Bone density 

It is striking that, based on bone density at the femoral neck, there is a threefold increase in risk of hip fracture 
in over one-half of our patients with premature ovarian failure. Hip fracture is associated with major morbidity. 
The dollar cost of hip fracture in the United States alone is more than $8 billion per year. In addition, there are 
human costs of pain, functional limitation, reduced quality of life, loss of independence, and inability to work. 
There is evidence that American women in general are experiencing more hip fractures earlier in life. In other 
words, the age-specific incidence of hip fracture has been increasing during the past three decades. The reasons 
for this age-specific increase are unknown, but increasingly sedentary life styles likely play a role. 

Even simple questions with tremendous public health import about osteoporosis go unanswered. For example, 
presently we do not know what type of weight bearing exercise is the most effective to increase bone density in 
young women. Our patients with premature ovarian failure are an ideal group in which to evaluate strategies to 
increase bone density and reduce risk of hip fracture. Patients with premature ovarian failure experience periods 
of hypoestrogenism before the disorder is recognized, and hypoestrogenism causes rapid loss of bone mineral 
density. Esfrogen replacement therapy will halt this rapid loss, but alone will not restore lost bone. A safe and 
effective method to restore bone density for these women is needed. One in 100 American women develop 
premature ovarian failure. 

b. Luteinized graafian follicles 

By identifying luteinization of graafian follicles as a major pathophysiologic mechanism in patients with 
premature ovarian failure, this program has provided key insight into ovarian follicle function in these patients. 
When viewed from a broader perspective, this finding has public health implications to human reproduction that 
loom large and demand further investigation. Development of luteinized graafian follicles in patients with 
premature ovarian failure may be only one extreme of a vast continuum of human reproductive pathophysiology. 

Our findings raise the distinct possibility that reproductive pathology in maturing women may result from the 
decline in primordial follicle number. Reduced negative feedback on LH rather than an intrinsic defect within 
the individual remaining primordial follicles may be the mechanism of fertility decline. With advancing age, 
along with this decline in follicle number, women experience a concomitant dramatic decline in fertility, increase 
in the rate of pregnancy wastage, and increase in births resulting from chromosomal nondisjunction (trisomy 21). 
This reproductive pathology has been traditionally ascribed to "ovarian aging," but the pathophysiologic 
mechanism of this deterioration in reproductive function has never been defined. 

Our findings suggest that the excess supply of primordial follicles present in normal women plays a critical role 
by providing a cohort of follicles to provide negative feedback. This negative feedback maintains LH in the 
normal range, and prevents inappropriate luteinization of developing graafian follicles. Women begin puberty 
with approximately 300,000 primordial follicles in their ovaries, but only about 400 of these follicles actually 
ovulate during a woman's reproductive life. Whether there is a physiologic need for this vast excess of primordial 
follicles has been a mystery. 

Subtly inadequate negative feedback upon LH, subtle enough to evade detection by available clinical measures, 
might impair normal dominant follicle function and egg maturation in older menstruating women. If true, 
sfrategies to restore normal negative feedback on LH might improve fertility, reduce pregnancy wastage, and 
possibly even improve oocyte function to reduce the incidence of chromosomal nondisjunction. 



ZOl HD 00633-05 DEB 

c. Autoimmune oophoritis 

Our findings highlight the fallacy of adding treatments into clinical practice based on anecdotal reports of success, 
and emphasize the need for controlled studies to evaluate therapies for this condition. Patients with premature 
ovarian failure often experience spontaneous remission. 

Anecdotal reports have suggested that high-dose, long-term prednisone therapy may be useful in treating 
autoimmune ovarian failure. However, prednisone, when used in high-dose for a long-term, has substantial side 
effects. These include aseptic necrosis of bone requiring major surgical intervention. Despite this risk, patients 
with premature ovarian failure are being treated based on this anecdotal evidence. We are aware of one patient 
with premature ovarian failure who developed aseptic necrosis of bone on high-dose, long-term prednisone 
therapy administered elsewhere. 

To make progress toward a better understanding of human autoimmune oophoritis will require an integrated 
approach, such as presented here, to combine clinical and basic laboratory investigation with use of appropriate 
animal models. A major obstacle to progress in autoimmune premature ovarian failure is the lack of an accurate 
diagnostic method to identify patients who develop ovarian failure by this mechanism. The need is great to define 
the pathophysiology of autoimmune ovarian failure, to develop accurate tests to identify the pathogenic 
mechanisms, and to develop specific efficient therapies to reverse these pathogenic mechanisms without the side 
effects that accompany generalized immunosuppression. 

We are narrowing the search for autoimmune oophoritis. Work by previous investigators has demonstrated that 
ovarian wedge biopsy has a low yield in diagnosing autoimmune oophoritis. Based on our findings it appears 
that ovarian biopsy targeted at developing antral follicles will also have low yield in diagnosing this condition. 

As practicing physicians become aware of our findings through peer review publications and book chapters, 
unnecessary health care expenditures related to premature ovarian failure should decline, and these funds will 
become available for other health care needs. Clinicians seeing patients with premature ovarian failure often order 
antiovarian antibody titers in an effort to diagnose autoimmune premature ovarian failure. Some also order 
lymphocyte phenotyping to determine if HLA DR3 is present. These tests are expensive, and, based on our 
findings, add nothing substantive to the evaluation. 

Murine post-thymectomy polyglandular autoimmunity 

Autoimmune diseases rank among the major medical problems of today's industrialized societies. Women are 
more susceptible to autoimmune diseases than men, and autoimmune diseases in children can significantly impair 
normal growth and development. Independent from their medical and social consequences, autoimmune diseases 
present fiindamental questions about the development and regulation of the immune system. What mechanisms 
permit the immune system to differentiate autoantigen from invading antigen? Murine post-thymectomy 
polyglandular autoimmunity is particularly appealing as a model to gain insight into this development and 
regulation. In this model, autoimmunity is induced by a specific perturbation to the immune system, without the 
need for administration of exogenous antigen. 

a. Inciting ovarian antigen 

The novel oocyte-specific gene we have identified just might be the gene that produces the inciting ovarian 
antigen in this murine model. This gene may allow us to development a sensitive and specific serum marker for 
human autoimmune ovarian failure. If we detect an analogous human gene, we can employ the analogous human 
recombinant protein in an assay to detect oocyte-specific human ovarian autoantibodies. There is precedence for 
this strategy. In murine post-thymectomy autoimmune gastritis the inciting antigen has been identified as the 
gastric proton pump. Interestingly, antibodies to the gastric proton pump are also a useful marker for autoimmune 
gastritis in humans. In many models of organ-specific autoimmunity a single protein antigen plays a key role 



€9 



ZOl HD 00633-05 DEB 

in initiating disease, and dramatic cellular and humoral responses to this antigen develop early in the process. 

b) Perturbing the neonatal immune system 

We may have uncovered the major pathophysiologic mechanism by which neonatal thymectomy induces 
polyglandular autoimmunity. Our findings challenge fundamental mechanisms that have been proposed regarding 
the development of the neonatal immune system. Furthermore, our findings provide a basis for evaluating and 
treating human autoimmune disorders from the perspective of abnormal neonatal development. 

Our studies in this model might prove basis for a clinical trial of IL-12 therapy for children with the candidiasis 
endocrinopathy syndrome (autoimmune polyglandular failure type 1). This is an enigmatic autosomal recessive 
syndrome in which children with impaired cellular immunity, a THl dependent function, nevertheless develop 
autoimmune polyglandular failure. 

Proposed Course: 

Basic research 

We will finish sequencing the novel murine oocyte-specific gene that we identified. To identify this gene, we 
screened a mouse ovarian lambda gt-11 cDNA expression library with hith-titer serum from mice with post- 
thymectomy autoimmune oophoritis. If we can sequence an analogous human gene, we will employ the 
analogous human recombinant protein to develop an assay for high-titer oocyte-specific autoantibodies in patients 
with premature ovarian failure. We will also evaluate the use of human recombinant zona pellucida 3 (ZP3) 
protein as a more specific test to determine if patients with premature ovarian failure have antibodies against the 
human zona pellucida (radio-immunoprecipitation assay). 

We will determine if our novel oocyte-specific gene produces the inciting antigen in murine post-thymectomy 
autoimmune oophoritis. We will do this by expressing this single autoantigen in the thymus using transgenic 
techniques. If this is indeed the inciting antigen, the expression of this protein in the thymus should induce 
specific tolerance. This should abrogate the development of autoimmune oophoritis in A/J mice thymectomized 
as neonates. This same approach has been used previously to confirm that one specific protein, the 13 subunit of 
H/K ATPase (gastric proton pump), is the inciting antigen in post-thymectomy autoimmune gastritis. 

We will extend our experiments in murine post-thymectomy polyglandular autoimmunity to further study the 
development and regulation of the immune system. If neonatal thymectomy induces disease by impairing the 
development of a Thl response, disease should not occur if we can cause the shift to the adult Thl dominant 
response to occur before the thymectomy is performed on day 4 of life. To test this hypothesis, we will 
administer IL-12 on day 2 and 3 of life, perform thymectomy on day 4 as usual, then determine if this treatment 
restores normal Thl:Th2 balance and ameliorates the autoimmune oophoritis despite thymectomy. 

We will also extend our experiments into the clinic by examining children with the candidiasis-endocrinopathy 
syndrome (autoimmune polyglandular failure type 1) for evidence of a similarly predominant TH2 response in 
peripheral blood lymphocytes. If in these children such an abnormality in the normal THI:TH2 profile is indeed 
present, this would raise the possibility of a subsequent clinical trial employing IL-12 as a means not only to treat 
the chronic candidiasis, but also to possibly prevent the development of polyglandular failure. 

Clinical research 



We will continue our controlled trial of alternate day prednisone therapy in the treatment of biopsy-proven 
autoimmune premature ovarian failure. This protocol tests the hypothesis that a lower risk therapy (alternate-day, 
lower dose, shorter-term prednisone) will induce remission of autoimmune ovarian failure. 



90 



ZOl HD 00633-05 DEB 

We will begin a randomized controlled trial to evaluate strategies to improve bone density in women with 
premature ovarian failure. We will compare programs of intensive home-based weight-bearing exercise 
specifically designed to maximally load the hips and spine. Change in bone mineral density at the femoral neck 
measured by dual energy x-ray absorptiometry will be the primary outcome parameter to be compared. 

We will extend our investigation of luteinization of graafian follicles in patients with premature ovarian failure. 
We hypothesize that by suppressing gonadotropins, and then stimulating follicle growth with human recombinant 
FSH, we may prevent premature luteinization of follicles and improve ovulation rates. By using an open trial 
design, this study will test the hypothesis that this treatment induces ovulation in 33% of patients. We learned 
in previous studies that women with premature ovarian failure, who have a serum estradiol greater than 50 pg/ml 
during one two month period of weekly sampling, have an 8.7% chance of having an ovulatory serum 
progesterone level during a second independent two month period of observation (upper 95% confidence limit 
32%). 

We will extend our clinical investigation to determine if luteinized graafian follicles may represent one extreme 
of a continuum of human reproductive pathophysiology. No therapy proven by prospective controlled study is 
available to women over 40 years old who have unexplained infertility. We hypothesize that in women over 40 
who have unexplained infertility, by suppressing serum LH and then stimulating follicle growth and development 
with human recombinant FSH, we may prevent subtle premature luteinization of follicles and improve pregnancy 
rates. This study will test this hypothesis by a randomized, double-blind, placebo controlled, crossover design 
study. We will use the luteinizing hormone releasing hormone analog D-Trp^ GnRH (D-Trp'^-Pro'-NEt-GnRH) 
to suppress serum LH, and recombinant human FSH to stimulate follicle growth. Women with three years of 
unexplained infertility have a per cycle fecundity of 0.01. This treatment would be a practical therapy if it could 
increase the per cycle fecundity to 0.15. We need to enroll 95 patients to detect this difference 90% of the time. 

We will investigate vascular endothelial growth factor as an early molecular marker for ovarian follicle 
luteinization. To develop new therapies for patients with abnormal ovarian follicle function requires an 
understanding of normal human ovarian function. Surprisingly, little is known about the local factors that effect 
and regulate human follicle luteinization. Inappropriate luteinization of graafian follicles could be a major 
pathophysiologic mechanism preventing normal follicle function in many reproductive disorders. 
Neovascularization is a major histologic component of luteinization, and therefore, vascular endothelial growth 
factor might be a useful early molecular marker. Thirty healthy women who will be undergoing reversal of 
surgical sterilization by minilaparotomy will be recruited. They will have their surgery timed to take place within 
a 4 day window expected to include the luteinizing hormone (LH) surge. The time of the surge will be 
determined from blood samples collected every 4 hours during the days preceding surgery. At the time of tubal 
surgery, the dominant ovarian follicle will be excised using microsurgical techniques. The follicle will be snap 
frozen, sectioned, and prepared with appropriate radio-labelled probes to detect message for vascular endothelial 
growth factor. Signal intensity in the theca and granulosa cells will be compared in follicles removed before and 
after the LH surge. We will also continue our protocol that permits us to aspirate granulosa cells, follicular fluid, 
and oocytes from the follicles of women undergoing sterilization. 

We will investigate a method of ovulation induction for patients with Stein Leventhal syndrome who are resistant 
to clomiphene citrate therapy. Ovarian follicles contain androgen receptors, and androgens may play a role in 
follicle atresia. The hyperandrogenism associated with Stein Leventhal syndrome might impair ovarian follicle 
development through a receptor-mediated mechanism. Flutamide, a non-steroidal anti-androgen, effectively blocks 
the action of androgen at the receptor level. We will test the hypothesis that blockade of androgen action with 
flutamide will permit ovulation in women previously resistant to clomiphene citrate alone. We will employ a 
randomized, double blind, and placebo controlled study design. We will study 50 patients and determine 
ovulation rates by serial progesterone levels. 

In a new area of investigation for this project, we will evaluate fat-suppressed magnetic resonance imaging (MRI) 
as a non-invasive method to detect lesions of endometriosis in patients with severe invasive endometriosis. 



9 



ZOl HD 00633-05 DEB 

Endometriosis is associated with infertility in women, and evidence suggests the presence of endometriosis may 
interfere with normal ovarian follicle growth and development. While not classified as a malignancy, severe 
invasive endometriosis can nevertheless invade bowel, bladder, and even lead to ureteral obstruction. Often the 
disorder involves prolonged medical treatment and repeated surgical interventions. All currently available 
conservative treatment strategies for endometriosis are, in essence, palliative. An accurate, non-invasive method 
to detect and monitor endometriosis would facilitate efforts to develop more effective conservative therapies. 
Twenty patients with severe invasive endometriosis will be recruited. After appropriate preoperative evaluation 
and MRI examination, patients will undergo surgery as indicated by their individual clinical situation. The size 
and location of endometrial implants in the surgical field will be noted. The sensitivity, specificity, and positive 
predictive value for the fat-saturated MRI technique to detect 4 mm lesions will be generated by standard 
statistical methods. 

Protocols: 



91-CH-127 Nelson Ovarian follicle function in patients with premature ovarian failure 

This screening protocol is the focal point of our clinical and basic research effort on premature ovarian failure. 
We use this enigmatic disorder as a springboard to gain insight into ovarian function not only by seeing patients, 
but also by employing animal models and basic molecular biology in collaboration with basic scientists. In so 
doing, we take ftill advantage of the unique environment provided by the NIH Clinical Center. The potential for 
a broad public health impact of such an approach should not be underestimated. We have recruited 51 of the 150 
patients approved for this protocol. From this protocol we have learned that one-half of patients with premature 
ovarian failure have follicles that function intermittently, and based on controlled studies, we have also found that 
patients with this disorder can undergo remission after treatment with placebo. This emphasizes the need for 
controlled studies to evaluate treatments for this disorder. 

92-CH-223 Nelson Autoimmune premature ovarian failure: a controlled trial of alternate-day 

prednisone therapy 

No therapy for infertile patients with premature ovarian failure has been proven effective by prospective controlled 
study. Anecdotal reports have suggested that high-dose, long-term prednisone therapy may be useful in treating 
autoimmune ovarian failure. However, prednisone, when used in high-dose for a long-term has substantial side 
effects, including aseptic necrosis of bone requiring major surgical intervention. Despite this risk, patients with 
premature ovarian failure are being treated based on this anecdotal evidence. We are aware of one patient with 
premature ovarian failure who developed aseptic necrosis of bone on high-dose, long-term prednisone therapy 
administered elsewhere. This protocol will test the hypothesis that a lower risk therapy (alternate-day, lower dose, 
shorter-term prednisone) will induce remission of autoimmune ovarian failure. The protocol will use a double- 
masked, placebo-controlled design. Patients will undergo ovarian biopsy by laparoscopy to confirm the presence 
of autoimmune oophoritis. Ovarian biopsy carries a mortality risk approximately equivalent to the risk women 
accept when they decide to undergo surgical sterilization. Successful outcome will be defined as a return of 
ovulation as determined by weekly serum progesterone levels. The hypothesis that short-term, alternate-day 
prednisone therapy restores ovulation will be tested with an equality of proportions test comparing the proportion 
of patients who ovulate during placebo with the proportion of patients who ovulate during prednisone therapy. 

92-CH-153 Anasti Aspiration of granulosa cells, follicular fluid, and oocytes in women 

undergoing sterilization 

To develop new therapies for patients with abnormal ovarian follicle function requires an understanding of normal 
human ovarian function. This protocol permits us to collect human ovarian follicle fluid, human granulosa cells, 
and human oocytes. This enables us to define the normal biochemical and cellular parameters related to normal 
ovarian follicle function and ovum maturation. We obtain these materials from women undergoing laparoscopic 
sterilization. To be eligible for this protocol, women must be referred to us by their personal physician after they 






ZOl HD 00633-05 DEB 

have independently decided to undergo laparoscopic sterilization. Aspiration of ovarian follicles during 
laparoscopic sterilization has negligible risk. As part of protocol 92-CH-223 (above) we have demonstrated that 
patients with premature ovarian failure develop graafian follicles that are inappropriately luteinized. We have 
moved ahead under this protocol to investigate the process of luteinization in normal women. We have shown 
that human follicle fluid TGFI3 (transforming growth factor B) is positively correlated with follicle fluid 
progesterone levels, a marker for luteinization. This suggests TGFI3 may have a role in human granulosa cell 
luteinization. 

92-CH-16 Anasti Flutamide in the treatment of Stein-Leventhal Syndrome 

Anovulation associated with Stein Leventhal Syndrome causes infertility. Clomiphene citrate therapy is the first 
line of treatment for women with this condition who desire fertility. However, in some patients clomiphene 
citrate fails to induce ovulation. Presently, we have no effective therapy for patients with who fail to respond 
to clomiphene. Ovarian follicles contain androgen receptors, and the hyperandrogenism associated with Stein 
Leventhal Syndrome might impair follicular development through a receptor-mediated mechanism. Flutamide, 
a non-steroidal anti-androgen, effectively blocks the action of androgen at the receptor level. This protocol tests 
the hypothesis that blockade of androgen action with flutamide will allow patients to ovulate even thought they 
were previously resistant to clomiphene therapy alone. We employ a randomized, double blind, and placebo 
controlled study design. We plan to study 50 patients and determine ovulation rates by serial progesterone levels. 
Recruitment has been slow, and we plan to step up the effort on this protocol during the coming year. 



93-039 Nair Immunogenetics of autoimmune oophoritis in the neonatally thymectomized mouse 

model 

93-032 Nelson Autoimmune ovarian failure (murine) 



Publications 

Anasti IN, Flack MR, Froehlich J, Nelson LM. The use of human recombinant gonadotropin receptors to search 
for IgG mediated premature ovarian failure, J Clin Endocrinol Metab 1995;80:824-8. 

Anasti JN, Flack MR, Froelich J, Nelson LM, Nisula B. A potential novel mechanism for precocious puberty 
in juvenile hypothyroidism, J Clin Endocrinol Metab 1995;80:276-9. 

Anasti JN, Kimzey LM, Defensor RA, White B, Nelson LM. A controlled study of danazol for the treatment of 
karyotypically normal spontaneous premature ovarian failure, Fertil Steril 1994;62:726-30. 

Nair S, Mastorakos G, Raj S, Nelson LM. Murine experimental autoimmune oophoritis develops independently 
of gonadotropin stimulation and is primarily localized in the stroma and theca. Am J Reprod Immunol, in press. 

Nelson LM, Anasti JN, Flack MR. Premature ovarian failure. In: Adashi EY, Rock JA, Rosenwaks Z, eds. 
Reproductive endocrinology, surgery, and technology. New York: Raven Press, in press. 

Nelson LM, Anasti JN, Kimzey LM, Defensor RA, Lipetz KJ, White BJ, Shawker TH, Merino MJ. Development 
of luteinized Graafian follicles in patients with karyotypically normal spontaneous premature ovarian failure, J 
Clin Endocrinol Metab 1994;79:1470-5. 

Powell CM, Taggart RT, Drumheller T, Nelson L, Wangsa D, Wliite BJ: Molecular and cytogenetic studies of 
the critical region for premature ovarian failure. Am J Med Genet 1994;52:19-26. 



9. 



ZOl HD 00633-05 DEB 

Raj S, Nair S, Mastorakos G, Nelson LM. Anamnestic development of lymphocytic infiltration in murine 
experimental autoimmune oophoritis is primarily localized in the stroma and theca, Am J Reprod Immunol, in 
press. 

Tong Z-B, Nelson LM, Dean J. Inhibition of zona pellucida gene expression by antisense oligonucleotides injected 
into mouse oocytes, J Biol Chem 1995;270:849-53. 



3^ 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00634-03 DEB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or /ess. T/Y/e must fit on one line between the borders.) 

Metabolic Effects of Insulin-like Growth Factor I in Normal and Diabetic Adolescents. 



PRINCIPAL INVESTIGATOR lUst other professional personnel below the Principal Investigator.) IName, title, laboratory, and institute affiliation) 

P.I. C.A. Bondy Senior Clinical Investigator SGM, DEB, NICHD 



Others: 



Mark Bach 
Edward Chin 
Rick Reinhardt 



Senior Staff Fellow 
Senior Staff Fellow 
Medical Staff Fellow 



SGM, DEB, NICHD 
SGM, DEB, NICHD 
SME, DEB, NICHD 



COOPERATING UNITS lif any) 

None 



LAB/BRANCH 

Developmental Endocrinology Branch 



SECTION 

Section on Growth and Metabolism 



INSTITUTE AND LOCATION 



National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

1.0 



PROFESSIONAL: 

1.0 



OTHER: 

0.0 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects E (b) Human tissues 
E (a1) Minors 
D (a2) Interviews 



n (c) Neither 



SUMMARY OF Vi/ORK lUse standard unreduced type. Do not exceed the space provided.) 

This project has been terminated. 



PHS 6040 (Rev. 5/92) 



9i 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00636-03 DEB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT ISO characters or less. Title must fit on one line between the borders.) 

Endocrinology of Reproduction in Women 



PRINCIPAL INVESTIGATOR lUst other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

P.I. James H. Segars Sr. Clinical Investigator UMMR, SGR, DEB, NICHD 



Others: Susan Ntmez 
Gary Leong 
Domenica Rubino 
Paul Driggers 



Clinical Associate 
Visiting Associate 
Clinical Associate 
Staff Fellow 



UMMR, SGR, DEB, NICHD 
UMMR, SGR, DEB, NICHD 
UMMR, SGR, DEB, NICHD 
UMMR, SGR, DEB, NICHD 



COOPERATING UNITS lif any) 

K. Ozato, SMGI, Laboratory of Molecular Growth Regulation, NICHD; 
L. Nieman, SGR, Developmental Endocrinology Branch, NICHD 



LAB/BRANCH 



Developmental Endocrinology Branch 



SECTION 



Section on Gynecologic Research, Unit on Molecular Mechanism of Reproduction 



INSTITUTE AND LOCATION 

National Institute of Child Health and Human Development, NIH, Bethesda, MD 



TOTAL STAFF YEARS: 

4.0 



PROFESSIONAL: 

4.0 



OTHER: 





CHECK APPROPRIATE BOXIES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



(b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The broad objective of this project is to gain further understanding regarding the endocrinology of hormonal 
responses in women in order to provide rational design of therapies directed toward the clinical problems of 
endometriosis, pregnancy loss, fertility, and estrogen-dependent reproductive neoplasms. Advances during the current 
period include: 1) isolation of a novel RXR-binding protein from a breast cancer cDNA expression library; 2) 
demonstration that the novel RXR binding protein is present in breast cancer tissues and binds to the retinoid X 
receptor; 3) determination that mRNA for the novel protein is expressed in breast cancer cells in addition to other 
reproductive tissues; 4) demonstration that the novel protein and RXR interact in vivo using the dual hybrid yeast 
system; 5) demonstration that the retinoid X receptor interacts with the general transcription factor TFIIB in a yeast 
system; 6) determination of the regions required for RXR binding to TFIIB using GST binding assays and co- 
immunoprecipitation studies; 7) a demonstration that the interaction between RXR and the general transcription factor 
IIB requires ligand, and 8) demonstration of RXR activation of estrogen responsive genes in the presence of the 
perxisome proliferator-activated receptor (PPAR). We plan to further characterize the novel RXR-binding protein and 
study additional proteins that may contribute to modulation of estrogen responsive genes. 



PHS 6040 (Rev. 5/92) 



9 



u 



ZOl HD 00636-03 DEB 

PROJECT DESCRIPTION : 

OBJECTIVE : The overall objective remains unchanged during the current period. Our objective continues to be 
to gain a further understanding of the role of the retinoid X receptor in the modulation of estrogen responsive genes. 
This objective is approached in order to understand the mechanisms involved in tissue specific regulation of nuclear 
hormone receptor function. As proposed, research during the current period focused on the study of proteins other 
than the estrogen receptor which may contribute to regulation of estrogen responsive tissues. In addition we tested 
naturally occurring estrogen responsive promoters by regulation of the retinoid X receptor and PPAR. Lastly, use 
of the dual hybrid yeast system has begun to permit characterization, isolation, and functional study of RXR action 
and modulation of the receptor action by RXR-binding proteins. During the current period we have: 1) continued 
to characterize the role of the PPAR modulation of estrogen responsive genes; 2) isolated a novel RXR binding 
protein from a breast cancer cDNA library; 3) demonstrated that the retinoid X receptor functions in part by binding 
to the general transcription factor TFIIB. 

METHODS : 

Cotransfection assays were used to test for modulation of estrogen responsive genes by nuclear hormone 
receptors. Binding of receptors was tested using baculovirus and bacterial expressed proteins in addition to in vitro 
translated receptors. Further, a DNA receptor precipitation assay was developed which permitted a demonstration 
of RXR receptor mutant binding to estrogen responsive elements. 

Further, using a method of non-radioactive detection of receptor protein complexes from lambda phage 
expression library, a protein binding to the retinoid X receptor was identified. Far Western analysis was also used 
to test binding of this protein to the retinoid X receptor and to proteins present in nuclear extracts prepared from 
breast cancer cells. 

In addition to the above assays, levels of receptor were studied with standard Western, Northern, Southern, 
and mobility shift analysis. 

MAJOR FINDINGS : 

During the period we found that nuclear extracts prepared from breast cancer ceils express several RXR 
binding proteins with varied molecular weights of approximately a 160, 140, 120, 70 and 30 KD molecular weight. 
Using an expression library prepared from breast cancer mRNA and the non-radioactive detection method for 
receptor protein interaction described last period, we isolated a cDNA clone expressing a protein which bound to 
the retinoid X receptor. Analysis from the published sequences present in the gene sequence data banks show the 
gene to be novel. Further, binding studies using both in vitro translated proteins as well as baculovirus expressed 
proteins confirmed the binding of the protein to the RXR receptor expressed in recombinant form. In addition 
Northern analysis confirmed expression of the mRNA in breast cancer tissues as well as other reproductive tissues. 
Mapping studies confirmed specific regions of the novel protein were required for binding to the retinoid X receptor. 
Using serial screening of library and overlapping clones, the entire cDNA encoding the mRNA was isolated. 

Further, the retinoid X receptor was expressed in yeast using the dual hybrid yeast system. Analysis 
confirmed activation of the receptor which was ligand dependent. Further analysis showed evidence of binding to 
the newly characterized novel RXR binding protein. This suggests that the interaction between the newly identified 
protein species and RXR can occur in vivo. 

In a related project, the function of RXR was studied in detail using the yeast dual hybrid system. RXR 
was noted to interact with the general transcription factor, TFIIB, in a ligand dependent fashion. Cotransfection 
assays performed in P-I9 embryonal carcinoma cell lines confirmed activation in undifferentiated cells as well. 
Using GST binding studies and coprecipitation studies, the regions required for interaction between the basal 



97 



ZOl HD 00636-03 DEB 

transcription factor, TFIIB and the retinoid X receptor were identified and characterized. It was determined that 
the carboxyl region of RXR is required for binding to the transcription factor TFIIB. Furthermore, a detailed 
analysis using both functional and binding studies revealed that, while binding was necessary for TFIIB-dependent 
activation, binding alone was not sufficient for RXR to increase reporter activity. This observation suggests an 
additional requirement for activation by the transcription factor TFIIB in addition to receptor binding. 

In addition to the above findings, studies have been performed with the peroxisome proliferator activated 
receptor. These studies have shown that the PPAR RXR heterodimeric pair noted (in prior reports) is able to bind 
a naturally occurring estrogen response element, specifically of that present in the oxytocin gene. Furthermore, 
cotransfection studies have demonstrated that addition of RXR and PPAR confer a ligand dependent responsiveness 
to this naturally occurring promoter. 

SIGNIFICANCE TO BIOMEDICAL RESEARCH IN THE PROGRAM OF THE INSTITUTE : 

A characteristic of reproductive tissues is their ability to respond to steroid hormones, however, it is not 
known how signals for these hormones are modulated at the tissue level. That is, addition of the anti-estrogen 
tamoxifen works as a anti-estrogen at the level of the breast, however, it is stimulatory to the endometrium. This 
paradox has puzzled reproductive scientists. Since we have previously shown that the retinoid X receptor was 
capable of modulating estrogen responsive genes, we have tested the hypothesis that the retinoid X receptor may 
be modulated at the tissue specific level by specific factors expressed in reproductive tissues. To identify such 
factors we have used a protein interaction cloning strategy to isolate a novel RXR binding protein present in breast 
cancer tissues. Breast cancer is a major public health concern that will affect 1 in 9 women. In addition to 
relevance to breast cancer, the present work contributes to an understanding of the modulation of nuclear hormone 
receptor function at the level of reproductive tissues. Thus the work may have relevance beyond a specific tumor 
and may be of more generalized significance. 

Further, an understanding of the function of the retinoid X receptor is inherently interesting since little is 
known about the relevance of gene regulation by this receptor to reproductive function. 

The demonstration that the retinoid X receptor is capable of binding to the transcription factor TFIIB 
furthers knowledge regarding receptor-mediated gene activation. In addition to previously published methods of 
testing for receptor interaction with basal transcription factors, we have used the dual hybrid yeast system which 
permits testing of the significance of this interaction in vivo. The in vivo demonstration of a receptor interaction 
with a general transcription factor in the yeast cells is unique. Our findings suggest that the retinoid X receptor is 
capable of interacting with other transcription factors present in yeast which can compete for interaction with TFIIB. 
Further, the demonstration of the interaction of the retinoid X receptor with TFIIB suggests that (since RXR 
contributes to heterodimeric interactions of receptors on particular hormone response elements) in some cases both 
members of a heterodimeric receptor pair are able to interact with the basal transcription complex via TFIIB. Thus 
RXR may not simply function to increase or augment DNA binding, but rather contribute to activation of basal 
transcription machinery. In addition, expression of the retinoid X receptor in yeast permits functional analysis in 
a system devoid of endogenous retinoid X receptor enabling our group to clearly define the regions of the retinoid 
X receptor involved in interaction with other hormone receptors as well as with other interacting proteins and basal 
transcription factors. Additional studies are required to further define receptor interactions using this system. 

PROPOSED COURSE : 

We plan to continue to study and characterize the novel RXR binding protein which is expressed in 
reproductive tissues. Specifically, we will determine the nature of this protein, the possible relevance of this protein 
to oncogenesis in reproductive tissues, and further characterize the regions and domains of this novel protein with 
regard to function. In addition, we plan to test naturally occurring estrogen responsive and retinoid X responsive 
genes for regulation by this novel receptor binding protein. Further, we plan to study endometrium and reproductive 






ZOl HD 00636-03 DEB 

tissues for expression of this novel retinoid X receptor binding protein in an effort to link expression with function. 

Our second aim is to continue to study the specific function of the retinoid X receptors in reproductive 
tissues. To accomplish this goal we will continue to utilize the dual hybrid yeast system to characterize the function 
of the retinoid X receptor in a system devoid of endogenous RXR expression. Additional efforts will be made to 
understand the factors that modulate expression of the retinoid X receptor in reproductive tissues. 

PUBLICATIONS : 

Chudacoff R, Alexander J, Alvero R, Segars JH. A modified method of tissue expansion vaginoplasty for treatment 
of congenital vaginal agenesis, Obstetrics and Gynecology, in press. 

Leong G, Blanco JG, Rolfes R, Marton M, Wang K, Wang IM, Moehle C, Tan A, Ozato K, Segars JH. Examination 
of the interaction between the retinoid X receptor and the general transcription factor IIB (TFIIB) in the dual hybrid 
yeast system. Endocrinology under 35, in press. 

Nunez SB, Medin JA, Keller H, Wang K, Ozato K, Whali W, Segars JH. Retinoid X receptor P and the peroxisome 
proliferator-activated receptor activate an estrogen response element, Rec Prog Horm Res 1995;50:409. 



9li 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD-00637-02 DEB 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or /ess. Title must fit on one line between the borders.! 

Steroid Hormone Action in Female Reproduction 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

P.I. L.K. Nieman Research Medical Officer URM, SGR, DEB, NICHD 



Others: 



T. Rarick 
J. Segars 
T. Kim 
P. Driggers 
M. Passaro 



Medical Staff Fellow 
Sr. Clinical Investigator 
Special Volunteer 
Senior Staff Fellow 
Clinical Associate 



LTPB, NICHD 
MMTP, SGR, DEB, NICHD 
URM, SGR, DEB, NICHD 
MMTP, DEB, NICHD 
SME, DEB, NICHD 



COOPERATING UNITS (if any) 

Maria Merino, M.D., Clinical Pathology, NICHD; Nancy Alexander, Ph.D., Contraceptive Development Branch, 
NICHD 



LAB/BRANCH 



Developmental Endocrinology Branch 



SECTION 



Section on Gynecologic Research, Unit on Reproductive Medicine 



INSTITUTE AND LOCATION 

National Institute of Child Health and Human Development 



TOTAL STAFF YEARS: 

1.0 



PROFESSIONAL: 

1.0 



OTHER: 





CHECK APPROPRIATE BOX(ES) 

(a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



H (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The purpose of this project is to understand better the development of the endometrium and oocyte in women and to 
investigate the role of gonadal steroids in these processes. Studies evaluating ovarian function in women suggested 
that growth hormone (GH) may be an important mediator of follicular development, as its receptor is present in the 
granulosa cells of the developing follicle and in the corpus luteum, the estrogen and progesterone steroidogenic 
compartments. We also demonstrated that a complete fast for 72 hours during the mid-follicular phase does not 
disrupt folliculogenesis. ovulation, or menstrual cycle dynamics in normal weight women, suggesting that brief 
periods of undernutrition are not deleterious to reproductive function. Another study evaluated the effect of aging on 
reproductive function. FSH was increased, and inhibin decreased, in the luteal-follicular transition of women aged 40 
- 50 years compared to those aged 20 - 30 years, but the incidence of abnormal endometrial biopsies was similar. 
These results suggest that follicular recruitment, but not luteal fiinction or endometrial maturation, is disturbed in 
cycling women over 40 years old and may contribute to decreased fertility. 

The process of uterine remodeling that is so remarkable in the mammalian uterus is achieved through coordinated 
proliferation, differentiation and cell death. While the gonadal steroids estradiol and progesterone appear to be 
required for these organ-specific processes, the mechanism(s) by which these processes are regulated remain unclear. 
Potential mediators of estrogen action include IGF-1 and c-Myc, a transcription factor known to be induced by 
growth factors. We used a well-characterized in vivo ovariectomized mouse model to correlate sex steroid-induced 
proliferation with the induction of IGF-1 and c-Myc mRNA in the uterine endometrium. We have found that lGF-1 
and c-Myc levels parallel each other and the estrogen-induced processes of differentiation in this model, suggesting 
that they are important to proliferation. The induction of IGF-1 by GH in other tissues prompted an evaluation of 
whether the GH receptor was present in the endometrium, based on the hypothesis that GH might act directly on the 
endometrium and so modulate estrogen effect. GH receptor was found to be present throughout the endometrium, 
but was not regulated by estradiol. 



PHS 6040 (Rev. 5/921 



100 



ZOl HD 00637-02 DEB 



Project Description: 

Objectives: 

Our long-term goal is to investigate infertility, to develop new contraceptives, and to evaluate new approaches 
to therapy of the menopause. Recent studies have sought to understand better the process of folliculogenesis 
and the role of growth hormone and IGF-1 in endometrial growth and development. 

We have investigated the effects of nutrition and aging on the process of folliculogenesis. In men and 
monkeys, caloric deprivation adversely affects reproductive function. As many women severely limit caloric 
intake for weight reduction, a similar effect of fasting on folliculogenesis might have important implications 
for women desiring fertility. Also, many women desire fertility after the age of 40, yet fecundity decreases at 
that time. We investigated hormonal dynamics and endometrial morphology in older women to understand 
better this decline in reproductive ftinction. 

The process of uterine remodeling that is so remarkable in the mammalian uterus is achieved through 
coordinated proliferation, differentiation and cell death. While the gonadal steroids estradiol and progesterone 
are required for these organ-specific processes, the mechanism(s) by which these processes are regulated 
remain unclear. We postulate that IGF-1 and c-myc mediate estrogen's mitogenic effects and that growth 
hormone (GH) as well as estrogen might induce endometrial IGF-1 and mitogenesis. Observations that 
growth hormone increases uterine weight independent of estradiol in hypophysectomized rats and is associated 
with an increased incidence of fibroids in acromegalic women support this idea. We thus examined whether 
the GH receptor is present in estrogen treated murine uterus, and in fibroid tumors from women. 

Methods employed: 

Effects of age on luteal function and endometrial maturation: Thirty-two regularly cycling women aged 20 to 
30 or 40 to 50 years, had daily blood drawing starting on cycle day 6 to 10 and continuing until 2 days after 
the onset of next menses for LH, FSH, estradiol, inhibin, PPM and progesterone measurements. In addition, 
60 women, aged 20 to 30 or 40 to 50 years, had a total of 93 endometrial biopsies performed on day 7 to 9 
after the LH surge for histologic dating. 

Evaluation of the effects of fasting on folliculogenesis in women: Twelve women were randomly fed or 
fasted on cycle days 7 to 9 to investigate whether fasting for 72 hours might disrupt reproductive function in 
normally cycling women, as it does in men and monkeys. Gonadotropin secretion, follicle development by 
ultrasound, and follicular and luteal phase lengths were measured. 

GH receptor in normal ovaries and leiomyoma: Ovaries were obtained at the time of surgery from non- 
pregnant normally cycling women undergoing hysterectomy/oophorectomy for non-ovarian gynecological 
indications. Leiomyomata and surrounding myometria were obtained from women who did or did not receive 
preoperative GnRHa therapy. Tissues were processed for mRNA extraction and PCR detection of the GH 
receptor and GAPDH, and for in situ hybridization. 

Animal studies: An oophorectomized murine model was used to examine whether IGF-1 and c-myc might 
mediate gonadal steroid action in the uterine glands and stroma. In situ hybridization for IGF-1 and c-myc 
mRNA was correlated with mitotic index after direct Alzet pump infusion of IGF-1 into the comua, or after 
subcutaneous administration of RU 486, progesterone and/or 



lOi. 



ZOl HD 00637-02 DEB 



estrogen. A second study evaluated the presence of growth hormone receptor in this model using PCR and in 
situ hybridization. 

Major Findings: 

1. Effects of aging on folliculogenesis and endometrial development: Serum FSH levels were increased 
whereas inhibin concentrations were reduced in the luteal-follicular transition of women > 40 years. No other 
hormonal changes were seen in this population, including progesterone and PPM secretion. Disruption of 
endometrial maturation occurred at a similar frequency in both age groups. These results suggest that 
follicular recruitment, but not luteal ftinction or endometrial maturation, is disturbed in cycling women over 
40 years old and may contribute to the decline in fertility with aging. 

2. Evaluation of the effects of fasting on folliculogenesis in women: None of the outcome measures differed 
between the fed and fasted cycles. We conclude that the reproductive axis of normally cycling women of 
mature gynecologic age and > 20 % body fat is not adversely affected by a 3 day fast during the mid- 
follicular phase. These data contrast with the reported sensitivity of the hypothalamic-pituitary-gonadal axis 
to acute nutritional withdrawal in men and monkeys and may reflect different sensing of fuel availability in 
women, possibly mediated through a higher percent body fat. 

3. Ovarian production of GH receptor: PCR and in situ hybridization confirmed the presence of GH receptor 
mRNA in corpora lutea and granulosa cells of antral follicles, but not in stroma, theca cells or primordial 
follicles of 7 surgically-removed ovaries from normally cycling women. Interestingly, not all follicles stained 
for GH receptor, suggesting that its expression may be associated with specific physiologic states, such as 
active steroidogenesis, and be absent in others, such as atresia. 

4. Presence of GH receptor in leiomyoma: GH receptor mRNA was present in both leiomyoma and 
surrounding myometrium with greater expression in leiomyoma, suggesting that GH may act directly on the 
human uterus. GnRHa treatment reduced GH receptor gene expression, implying that GH receptor expression 
in leiomyoma may be regulated by sex steroids. 

5. Animal studies: After gonadal steroid freatment, endometrial IGF-1 and c-myc mRNA expression 
increased, and paralleled the pattern of differentiation and proliferation. Treatment with estrogen (alone and 
with progesterone and RU 486) induced proliferation of the epithelial cells, while the addition of progesterone 
shifted mitosis to the sfroma and resulted in differentiation of the epithelium. IGF-1 and c-myc showed 
coordinate expression in the epithelium (estrogen treatment) or both compartments (estrogen and progesterone 
treatment). The c-myc expression after IGF-1 alone was similar to that seen with estrogen. These 
preliminary findings suggest a role for IGF-1 as an estromedin. 

PCR and in situ hybridization demonstrated GH receptor in the murine endometrium. There was no specific 
regulation by estradiol, suggesting that GH receptor expression is constitutive and is not involved in 
modulation of gonadal steroid action. 

Significance to Biomedical Research and the Program of the Institute: 

These studies reflect the NICHD mission to understand better the physiology of normal and abnormal 
reproductive states and to develop contraceptive agents. The ability of the uterus to undergo coordinated 
timely growth and regression provides an excellent model to understand the influence of growth factors and 
protooncogenes on cell cycle progression and the way in which hyperplasia and uncontrolled cell growth is 
prevented in vivo. 



10^ 






ZOl HD 00637-02 DEB 



Proposed Course: 

1. Characterization of progesterone-dependent endometrial products. We hypothesize that progesterone- 
induced peptides are required for endometrial epithelial differentiation and hence, implantation. We will 
evaluate progesterone regulation of three candidate peptides: c-MET, the vitronectin receptor, and smooth 
muscle myosin II. Activation of the MET receptor by mesenchymally derived hepatic growth factor is an 
integral element of epithelial cell differentiation in other systems, but has not been examined in the uterus. 
The morphologic association of stroma and epithelium, and the coordinated growth of the endometrium after 
menses, suggest that hepatic growth factor may play a role in this process. The vitronectin receptor, 
comprised of the pair, avps, is an integrin that binds ligands on the trophoblast surface, and can be blocked 
with peptides containing the tripeptide ROD (Arg-Gly-Asp), which prevent implantation in the mouse. This 
integrin is present on epithelial cells on days 19 to 24 and is absent in up to 20% of infertile women with 
delayed mid-luteal endometrial development and endometriosis-associated infertility. Its regulation by 
progesterone is not known. Smooth muscle myosin II (SMMII) is expressed in glandular epithelial cells in 
the midluteal phase in the intact baboon, and is induced by estrogen and progesterone treatment in 
ovariectomized animals. We are unaware of studies of this peptide in human endometrium. 

Two experimental models will be used: in vitro culture of Ishikawa cells, an hormonally-responsive 
endometrial epithelial cell line, and examination of endometrial biopsies obtained from normally cycling 
women. 

Ishikawa cells will be grown in standard media containing fetal calf serum. To examine the effects of 
progesterone, estrogen, and antiprogestins on the expression of the candidate peptides, cells will be grown in 
media containing physiologic amounts of these compounds (estradiol 1 50 pg/mL; progesterone 20 pg/mL; RU 
486 20 pg/mL) for 48 hours, and then harvested and processed for in situ hybridization or 
immunohistochemistry. To evaluate progesterone induction of other peptides, we will use mRNA from these 
treatment groups and the technique of differential display to identify potential progesterone-dependent 
epithelial products without ascertainment bias. 

Normally cycling women will be recruited to undergo an endomefrial biopsy, blood sampling and ovarian 
ultrasound. All cycles will be characterized hormonally by determination of the LH surge and by 
measurement of estradiol and progesterone. Follicular phase biopsies will be correlated with the size of the 
dominant follicle by ultrasound. Luteal phase biopsies will be timed according to the LH surge and will be 
obtained using a Pipelle curette. A part of each specimen will be placed in formalin for dating by Noyes' 
criteria. The remaining tissue will be used for study procedures, including in situ hybridization or 
immunohistochemistry to localize the candidate peptides to epithelial and/or stromal cells and to correlate 
changing patterns of expression over the menstrual cycle with hormone levels. The DNA sequence of the 
candidate peptides is known, and standards and antibodies to each are in use in our laboratory or available to 
us. 

2. The potential contraceptive action of low dose antiprogestins: We previously showed that RU 486 can 
retard endometrial maturation in women and inhibit implantation in the guinea pig, suggesting that 
antiprogestins may be effective contraceptive agents that render the endometrium hostile to implantation 
without affecting hormonal rhythm. Further development of these properties of antiprogestins would 
represent a significant new advance in contraceptive technology. 

RU 486 is no longer available from Roussel-UCLAF. However, the Contraceptive Development Branch, 
NICHD, has another antiprogestin, CDB 2914. Results from pre-clinical studies suggest that its activity is 
similar to that of RU 486. We will develop this compound for extramural and intramural clinical trials, 
beginning with protocol 95-CH-0168, "Dose-response Relationships for the Antiprogestin CDB 2914 in 



10< 



ZOl HD 00637-02 DEB 



Cycling Women". Women will receive the compound in the luteal phase in this phase 1-2 trial, to determine 
safety and to provide initial information about biologic effects. Blood will be obtained for pharmacokinetic 
studies, and for evaluation of changes in Cortisol, ACTH, LH, FSH, estradiol and progesterone. Women will 
chart menses and basal body temperature to evaluate antagonism of endometrial or hypothalamic progesterone 
action. Should the compound appear promising, another protocol will be proposed to include endometrial 
biopsy, which will provide placebo or antiprogestin-exposed tissue to test whether endometrial candidate 
peptides are regulated by progesterone. 

Publications: 



Batista MC, Cartledge TP, Zellmer AW, Merino MJ, Axiotis C, Bremner WJ, Nieman L. Effects of aging on 
menstrual cycle hormones and endometrial maturation, Fertil Steril 1995;64:492-299. 

Nieman LK. Estrogens and progestins. In: C Smith & A Reynard, eds. Essentials of Pharmacology. 
Philadelphia:WB Saunders, 1995;563-573. 

Olson BR, Cartledge T, Sebring N, Defensor R, Nieman LK. A three day fast during the mid-follicular phase 
does not disrupt reproductive fiinction of normal-weight sedentary women, J Clin Endocrinol Metab 
1995;80:1187-93. 

Sharara FI & Nieman LK. Growth hormone receptor mRNA expression in leiomyoma and surrounding 
myometrium, Am J Obstet Gynecol, in press. 

Sharara FI & Nieman LK. Identification and cellular localization of growth hormone receptor gene 
expression in the human ovary, J Clin Endocrinol Metab 1994;79:670-672. 

Sharara FI, Bhartiya D, Nieman LK. Growth hormone receptor gene expression in the mouse uterus: 
modulation by gonadal steroids, J Soc Gynecol Invest 1995;1:285-9. 

Patents: 

none 



10^ 



f 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD-00638-02 DEB 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT {80 characters or less. Title must fit on or}e line between the borders.) 



The Physiology of Hypercortisolism 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

P.I. L.K. Nieman Research Medical Officer URM, SGR, DEB, NICHD 



Others: J. Yanovski 

D. Papanicolaou 
G. Chrousos 
G. Cutler, Jr. 
M. Magiakou 



Supervisory Medical Officer, Pediatrics 

Clinical Associate 

Chief, SPE, DEB, NICHD 

Chief, SDE, DEB, NICHD 

Special Volunteer 



SDE, DEB, NICHD 
SPE, DEB, NICHD 
SPE, DEB, NICHD 
SDE, DEB, NICHD 
SPE, DEB, NICHD 



COOPERATING UNITS lif any) 

John Doppman, Department of Radiology, CC; Edward Oldfield, SND, NINDS 



LAB/BRANCH 



Developmental Endocrinology Branch 



SECTION 



Section on Gynecologic Research, Unit on Reproductive Medicine 



INSTITUTE AND LOCATION 

National Institute of Child Health and Human Development 



TOTAL STAFF YEARS: 

1.5 



PROFESSIONAL: 
1.5 



OTHER: 





CHECK APPROPRIATE BOXIES) 

(a) Human subjects D (b) Human tissues 
D (a1) Minors 
D (a2) Interviews 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Cushing syndrome, a fatal disease, is suspected in many thousands of patients each year, but confirmed in only a 
traction of these. This project seeks to identify accurately which patients have Cushing syndrome, to define the 
etiology of their disease and to treat it optimally. 

A major initiative in the past year has been to improve the approach to the differential diagnosis of Cushing 
syndrome. This effort included development of optimal criteria for interpretation of diagnostic tests that maintain 
100% specificity for the diagnosis of Cushing's disease. We compared the performance of the 8 mg overnight 
dexamethasone suppression test in 41 patients who also received the traditional 6 day test and identified the best 
timepoints at which to measure Cortisol for optimal diagnostic accuracy. We achieved 65 - 70% sensitivity using 
refined criteria for the traditional and overnight tests, and a 91% sensitivity if the tests were combined. Similar 
approach was used to optimize the traditional 4 day metyrapone stimulation test to develop criteria for 100% 
specificity in 185 patients with ACTH-dependent Cushing's syndrome. This test yielded a sensitivity of 72% using 
urine 17-hydroxysteroid and plasma 1 1-deoxycortisol endpoints. When combined with the traditional dexamethasone 
suppression test, sensitivity increased to 88%. These studies indicated that tests for the differential diagnosis of 
Cushing's syndrome can be combined to give improved sensitivity at 100% specificity. Further work will define an 
optimal cost-effective diagnostic strategy. In response to a report that venous sampling from the cavernous sinuses is 
superior to CRH-stimulated inferior petrosal sinus sampling, we also evaluated this test in 15 patients. In our hands, 
cavernous sinus sampling failed to diagnose correctly 20% of patients, while inferior petrosal sinus sampling 
correctly identified Cushing's disease in all patients. 



PHS 6040 (Rev. 5/92) 



10^ 



o 



ZOl HD 00638-02 DEB 



Project Description 

Objectives: 

This project seeks to identify accurately which patients have Cushing's syndrome, to define the etiology of their 
disease, and to treat it optimally. Cushing's syndrome, a fatal disease, is suspected in many thousands of patients 
each year, but confirmed in only a fraction of these. Once identified, its cause must be found and successfully 
treated. The identification, differential diagnosis and treatment of this syndrome pose problems for the clinician. 

There is no test that reliably separates individuals with pseudo-Cushing's states, who have mild hypercortisolism 
and minimal physical features of Cushing's syndrome, from those with mild or intermittent Cushing's syndrome, 
who may present in an identical fashion. We are exploiting the different pathophysiology in the pseudo-Cushing's 
and Cushing's syndrome states to develop new tests for this distinction. Patients with Cushing's syndrome have 
either an ACTH-dependent abnormality (corticotrope adenoma or ectopic ACTH-secreting tumor), or a primary 
adrenal tumor; in this setting the CRH neuron is appropriately suppressed. In contrast, patients with pseudo- 
Cushing's syndrome have an hypothalamic abnormality, hypersecretion of CRH; the pituitary and adrenal glands 
are normal. As a result of these differences, we hypothesize that patients with pseudo-Cushing's syndrome might 
maintain a normal diurnal pattern of Cortisol secretion, in distinction to the invariant Cortisol levels seen in 
patients with Cushing's syndrome. 

Our second initiative is to improve the approach to the differential diagnosis and treatment of the causes of 
Cushing's syndrome. The differential diagnosis of Cushing's syndrome, especially the ACTH-dependent forms, 
is difficult. The treatment of ectopic ACTH secretion depends on localization and surgical removal of the tumor, 
which may be occult in up to 50% of cases. 

Methods employed: 

The methods used in these clinical investigations involve blood and urine collection for hormone measurements 
by RIA with and without administration of a variety of provocative agents. Standard radiographic studies and 
catheterization procedures are performed to identify a tumor. The diagnosis of pseudo-Cushing's syndrome is 
confirmed by long-term physical and biochemical evaluation that fails to demonstrate convincing evidence for 
evolving Cushing's syndrome. The diagnosis of Cushing's syndrome is confirmed by physical examination and 
standard biochemical criteria; an etiologic diagnosis is assigned only after surgical cure or histopathologic 
identification of an ACTH-containing or primary adrenal tumor. 

Major Findings: 

Differential Diagnosis of Cushing's svndrome. We continue to evaluate tests for the differential diagnosis of 
Cushing's syndrome. We compared the performance of the standard 6 day, 8 mg, dexamethasone suppression 
test with the 4 day metyrapone stimulation test and the 8 mg overnight dexamethasone suppression test. Our 
eventual aim is to develop a decision tree based on the individual and combined diagnostic accuracy of these and 
other tests (CRH stimulation and inferior petrosal sinus sampling). In response to a report that venous sampling 
from the cavernous sinuses is superior to CRH-stimulated inferior petrosal sinus sampling (J Clin Endocrinol 
Metab 76:637-641, 1993), we also evaluated this test. 

1. Overnight dexamethasone suppression test: We compared the performance of the 8 mg overnight 
dexamethasone suppression test in patients who also received the traditional 6 day test and identified the best 
timepoints at which to measure Cortisol for optimal diagnostic accuracy. 

Various pre- and post-dexamethasone time points were examined and the test was optimized as follows: blood 



lOu 



ZOl HD 00638-02 DEB 



is drawn for Cortisol determination at 0900h before and 0830h after administration of dexamethasone, 8 mg, at 
2300h. Using these time points, we found tiiat a > 69% suppression of plasma Cortisol yielded a 71% sensitivity 
and 100% specificity in 41 patients (34 Cushing's disease; 7 ectopic ACTH secretion). When compared to the 
criterion developed previously by Tyrrell et al. (Ann Intern Med 104:180, 1986; suppression of plasma Cortisol 
> 50% at time combination 0800h before and after dexamethasone), the sensitivity was 88%, but the specificity 
decreased to 57%. To achieve 100% specificity of the test with the 0800h sampling times, a > 80% suppression 
of plasma Cortisol was required in our patients, which dropped the test sensitivity to 59%). This study 
demonstrates the importance of evaluating a large number of patients so that comparison groups of appropriate 
size are available. A similar sensitivity and specificity (100%) were achieved with the standard 6 day test, using 
criteria of > 69% suppression of 1 7-hydroxysteroid excretion (sensitivity 68%) or > 90% suppression of urine 
Cortisol excretion (sensitivity, 65%). 

2. Metyrapone stimulation test: We compared the traditional 4 day metyrapone stimulation test to develop 
criteria for 100% specificity in 185 patients with ACTH-dependent Cushing's syndrome. We found that the test 
could be reduced to two days: the day before and during metyrapone administration (750 mg every 4 hours, for 
six doses, beginning at 0800h on the second day). Urine is collected on both days, beginning at 0800h, for 17- 
hydroxysteroid measurement. Plasma 11-deoxycortisol is measured at 0800 at and 24 hours (before 
metyrapone), and at 48 hours (after metyrapone), and the difference between the post- and the mean pre- 
metyrapone values is calculated. Optimal criteria for the diagnosis of Cushing's disease were a 400-fold increase 
in 11-deoxycortisol and a > 70% stimulation in 1 7-hydroxysteroid excretion. When used together, a positive 
response to either criterion yielded 72% sensitivity at 100% specificity. When combined with the standard 
dexamethasone suppression test, using the criteria above, the sensitivity increased to 88%. 

3. Cavernous sinus sampling vs. inferior petrosal sinus sampling: During a single sampling procedure, ACTH 
levels were compared in simultaneous samples obtained using tracker catheters inserted into the cavernous sinuses, 
and in simultaneous samples obtained from both petrosal sinuses before and after administration of CRH. 15 
patients were studied prior to surgical cure of Cushing's disease. We found that unstimulated levels of ACTH 
in the cavernous sinuses were higher than unstimulated levels of ACTH in the petrosal sinuses, but 3 of 15 
patients failed to show sufficient central-to-peripheral cavernous sinus ratios and thus were falsely negative for 
the diagnosis of Cushing's disease (test sensitivity, 80%). By comparison, the test sensitivity for the petrosal 
sinus samples was 87% for the unstimulated samples and 100% for the CRH-stimulated samples. Because of the 
20% false negative rate, we do not recommend that cavernous sinus sampling without CRH be used for the 
differential diagnosis of ACTH-dependent hypercortisolism. 

4. Pediatric Cushing's syndrome: We analyzed the clinical presentation, diagnostic evaluation, and treatment 
of 59 patients with Cushing's syndrome between the ages of 4 and 20 years, admitted to the NIH. Fifty had 
Cushing's disease, six had primary adrenal disease, and three had ectopic ACTH secretion. Of those with 
Cushing's disease, magnetic resonance imaging of the pituitary indicated a tumor in only 52%. Transsphenoidal 
surgery was curative in 48 of 49 who underwent transsphenoidal exploration. We showed that previously 
developed criteria for interpretation of the CRH test and inferior petrosal sinus sampling were useftil in children. 



Significance to Biomedical Research and the Program of the Institute: 

Development of improved tests for the diagnosis of Cushing's syndrome has a great potential impact on public 
health. In our experience, the greatest challenge in the evaluation of patients with possible Cushing's syndrome 
is to establish the diagnosis. Cushing's syndrome is considered, but is statistically improbable, in thousands of 
patients each year. The currently available tests do not minimize equivocal information and may lead to a 
mistaken diagnosis of Cushing's syndrome, which prompts further tests and places the patient at risk for 
inappropriate intervention, while increasing health care expenditure. Our previous finding that the classic 



10 



ZOl HD 00638-02 DEB 



screening test, UFC excretion, has a significant false negative and false positive rate illustrates the importance 
of continued efforts to improve both the sensitivity and the specificity of screening tests. 

The above effort to better discriminate pseudo-Cushing's syndrome may impact on tens of thousands of patients 
each year. In contrast, our effort to refine the approach to the differential diagnosis of Cushing's syndrome has 
a smaller overall benefit to the public health. However, we are now recognized as one of a handful of centers 
in the world with experience of more than 200 cases, and so we have a unique opportunity to develop an accurate, 
cost-effective diagnostic strategy that can be used by others with less experience. The benefits to the public 
health of this large ongoing experience are illustrated in the studies cited in this report. Many of the tests for the 
differential diagnosis of Cushing's syndrome have been developed in small groups of patients, with very few of 
the important comparison groups, such as those with ectopic ACTH secretion. Our ongoing analysis of a large 
group of patients has enabled us to simplify, standardize and set criteria for the interpretation of these tests. Also, 
by quickly evaluating reports advocating new tests based on small numbers of patients, we help to establish 
whether the new tests have value in a larger population. Similarly, by virtue of its unique size, our report of a 
large number of pediatric patients with Cushing's syndrome revealed that ectopic ACTH secretion must be 
considered in this population, and also showed the unrecognized utility of transsphenoidal surgery in these 
patients. 

Proposed Course: 

Diagnosis of Cushing's syndrome. We will continue to measure diurnal plasma Cortisol to assess its power to 
discriminate between pseudo-Cushing's syndrome and Cushing's syndrome. Although preliminary data indicate 
that this test may have a high diagnostic accuracy, it is not practical, so we plan to obtain salivary Cortisol 
measurements. We anticipate this change in methodology will improve the test's sensitivity, as hypercortisolism 
suppresses CBG and increases free Cortisol, the fraction measured in saliva. It also will make the test convenient 
and feasible for in-home use, further decreasing the cost of evaluation for Cushing's syndrome. We plan to 
extend our previous observation that dexamethasone pre-treatment suppresses the response to an CRH stimulation 
test by administering dexamethasone or placebo prior to inferior petrosal sinus sampling in normal individuals 
and those with pseudo-Cushing's or Cushing's syndrome. This will allow us to establish the range of basal and 
CRH-stimulated ACTH levels (with and without dexamethasone) in these groups; minimal overlap may suggest 
this as a diagnostic approach, especially for patients with mild hypercortisolism. Even when it is clear that these 
patients have Cushing's syndrome, the interpretation of differential diagnostic tests is suspect because of the 
concern that the corticotropes are inadequately suppressed. 

Differential Diagnosis of Cushing's syndrome. The availability of CRH and the development of IPSS and CRH 
testing has revolutionized the differential diagnosis of Cushing's syndrome, leading to near 100% sensitivity and 
specificity in our hands. It is unlikely, however, that IPSS will become widely available, since significant 
catheterization experience is necessary for its success. Additionally, it has a small, but measurable morbidity, 
and there is no consensus as to its appropriate role in the diagnostic strategy for Cushing's syndrome. We 
continue, therefore, to analyze the performance of all tests, with the goal of developing a simple and convenient 
decision tree for the evaluation of Cushing's syndrome that minimizes cost without compromising diagnostic 
accuracy. 

Because inferior petrosal sinus sampling is dependent on the skill of the radiologist, it is not widely available. 
Additionally, the morbidity of cerebrovascular accident attendant to the procedure is probably related to occlusion 
of small vessels. For these reasons, we are interested in exploring the diagnostic utility of jugular venous 
sampling for measurement of CRH-stimulated ACTH levels. We hypothesize that this procedure may have a 
diagnostic accuracy similar to that of inferior petrosal sinus sampling. It would have the advantage of being 
easier to perform, thus more widely available, and might also be more safe. We plan to compare the two 
procedures in 30 patients. 



10b 



ZOl HD 00638-02 DEB 



Unfortunately, the localization of ectopic ACTH-secreting tumors fails initially in 50% of these patients, and may 
not be achieved for 20 years, exposing them to the deleterious effects of hypercortisolism and the risk of 
metastatic disease. Thus, we plan to focus on promising venues for the localization of these tumors. We will 
evaluate the utility of radionuclide-labeled sandostatin, a somatostatin analog, for the detection of ectopic ACTH- 
secreting tumors, and will explore the feasibility of total body PET scan for identification of intrathoracic lesions. 

The rate of recurrence in patients successfully "cured" of Cushing's disease by transsphenoidal surgery has not 
been established, but may be as great as 10%. We continue to follow our cohort to determine the risk of 
recurrence in the group and are extending our earlier observation that the response to CRH in the post-operative 
period may predict those patients destined to recur. Should this be clearly established in a large group of patients, 
strong consideration may be given to early x-irradiation therapy in patients with abnormal post-operative CRH 
responses. 

Publications: 



Avgerinos PC, Yanovski JA, Oldfield EH, Nieman LK, Cutler GB Jr. The metyrapone and dexamethasone 
suppression tests for the differential diagnosis of Cushing's syndrome: a comparison, Ann IntMed 1994; 121:3 18- 

27. 

Dichek HL, Nieman LK, Oldfield EH, Pass HI, Malley JD, Cutler GB Jr. A comparison of the standard high- 
dose dexamethasone suppression test and the overnight 8-mg dexamethasone suppression test for the differential 
diagnosis of Cushing's syndrome, J Clin Endocrinol Metab 1994;78:418-422. 

Doppman JL, Nieman LK, Chang R, Yanovski J, Cutler GB Jr, Chrousos GP, Oldfield EH. Selective venous 
sampling from the cavernous sinuses is not a more reliable technique than sampling from the inferior petrosal 
sinuses in Cushing's syndrome, J Clin Endocrinol Metab 1995;80:2485-2489. 

Magiakou MA, Mastorakos G, Gomez MT, Doppman JL, Cutler GB Jr, Oldfield EH, Nieman LK, Chrousos GP. 
The NIH experience with Cushing's syndrome in children and adolescents: Presentation, diagnosis and therapy, 
N Engl J Med 1994;331:629-636. 

Nieman LK, Cutler GB Jr. Cushing's Syndrome. In: DeGroot L, ed. Textbook of Endocrinology. 
Philadelphia: WB Saunders, 1994; 1741-1 769. 

Patents: 

none 



lOu 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00841-13 BB 



PERIOD COVERED 

October 1, 1994 through September 



30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Methods for Comparing and Analyzing; Data from Several Complex Surveys 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.') (Name, title, laboratory, and institute affiliation) 

PI: K.F. Yu Mathematical Statistician BMSB, DESPR, NICHD 



Others: H.J. Hoffman 
M.D. Overpeck 
Y.J. Lee 



Chief, Epidemiology 

Epidemiologist 

Chief 



CD, EB, NIDCD 
EB, DESPR, NICHD 
BMSB, DESPR, NICHD 



COOPERATING UNITS fifanv) 

BB, CTS, DCPC, NCI (B. Graubard) ; BRB, NCI (E. Korn) 



LAB/BRANCH 

Biometry and Mathematical Statistics Branch, DESPR 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



-2_ 



L2_ 



TOTAL STAFF YEARS: 
0.0 



PROFESSIONAL: 
0.0 



OTHER: 
0.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human 
D (al) Minors 
D (a2) Interviews 



n (b) Human 



n (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 
This project has been terminated. 



PHS 6040 (Rev. 5/92) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00855-04 BB 



PERIOD COVERED 

October 1,1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Statistical Methods for the Common Odds Ratio of a Number of Contingency Tables 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
P.I.: K.F. Yu Mathematical Statistician BMSB, DESPR, NICHD 



Others: A. A. Herman 

J.F. Troendle 



Visiting Scientist 
Senior Staff Fellow 



EB, DESPR, NICHD 
BMSB, DESPR, NICHD 



COOPERATING UNITS fifanv) 



LAB/BRANCH 

Biometry and Mathematical Statistics Branch, DESPR 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
0.35 



PROFESSIONAL: 
0.35 



OTHER: 
0.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human 

n (al) Minors 

D (a2) Interviews 



n (b) Human 



C (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This Study will develop statistical methods for analyzing categorical data, 
particularly in the comparison of different groups . Examples are the common odds 
ratios for a number of contingency tables and the standardized risk difference in 
the comparison of two groups stratified by a covariate. The optimality properties 
of the developed methodology will be investigated and established. The likelihood 
equations are to be derived. The newly developed, maximum likelihood and 
conditional maximum likelihood methodologies are to be examined. The study has 
been extended to do a comparison of a number of contingency tables in the form of 
estimation and hypothesis testing. This methodology is particularly useful in 
detecting the adequacy of matching in the case-control epidemiological studies. 
All these methodologies have wide applications to epidemiological studies, 
biomedical studies, genetics and other disciplines. Examples include testing the 
Hardy -Weinberg Law across strata in genetics and comparing the black-white 
differences in birth specific infant mortality curves. 






PHS 6040 (Rev. 5/92) 



ZOl HD 00855-04 BB 



Pro.iect Description: 



Objectives: To develop statistical methods for estimating the odds ratio of a 
number of contingency tables and a family of standardized risk difference 
parameters. To develop statistical methods for testing hypotheses concerning a 
number of contingency tables. To study black-white differences in birth weight 
specific mortality curves. To study the Hardy-Wei nberg Law across strata. 

Methods Employed : Classical and modern statistical and probabilistic methods are 
employed to analyze the problems. New methodology is also developed to handle the 
problems. Computer and numerical techniques are utilized. 

Ma.ior Findings : An estimator proposed by Greenland and Holland (1991, Biometrics 
47, 319-322) for a standardized risk difference parameter is shown to be a maximum 
likelihood estimator if the consistent estimator of the common odds ratio is 
appropriately chosen. The statistical problem under consideration is 
reparameterized for a better understanding. Likelihood equations are derived. A 
family of easily computable estimators of the common odds ratio of a number of 
contingency tables is derived from a set of reasonable postulates. A necessary and 
sufficient condition has been discovered for the strong consistency of this family 
of estimators. Also, a strongly consistent estimator has been found for the 
asymptotic variance. Breslow's condition (1981, Biometrika 68:73-84) has been 
found to be faulty for the consistency of the Mantel Haenszel estimator for large 
sparse tables. This finding clarifies the confusion over this point in the vast 
literature. New methodology has been developed to study two sequences of 
probabilities. It is applied to study the black-white differences in birth weight 
specific mortality curves. It is also extended to study the Hardy-Wei nberg Law 
across strata. 

Significance to Biomedical Research and the Program of the Institute : Odds ratio 
and standardized risk difference parameters are commonly used parameters in many 
biomedical research and epidemiological studies. One example is the Better Babies 
Project in which the P.I. has participated. This study will enable better 
understanding and more efficient use of these parameters in substantive research 
programs. Testing procedures so developed provide more power in comparing black- 
white differences in birth weight specific mortality curves. 

Proposed Course of Pro.iect : Continuation of development of statistical methodology 
is planned. Statistical properties are to be investigated. Applications to 
substantive fields such as genetics will be further examined. 

Publications : 

Troendle JF, Yu KF. A note on testing the Hardy-Wei nberg Law across strata. Ann 
Hum Gen 1994;58:397-402. 

Jannarone RJ, Ma K, Yu KF, Gorman JW. Extended conjunctoid theory and 
implementation: A general model for machine cognition based on categorical data. 
Prog Neural Net 1995;3:361-425. 

Yu KF. A necessary and sufficient condition for the strong consistency of a family 
of estimators of the common odds ratio. Can J Stat 1995;23:215-25. 

Yu KF. A simple comparison of two sequences of probabilities. J Plan Inference 
1995. (In Press) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE 
NOTICE OF ENTTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00856-04 BB 



PERIOD COVERED 

October 1, 1994 throuRh September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.') 
Statistical Methods for a Mixture of Subpopulations 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

P.I.: K.F. Yu Mathematical Statistician BMSB, DESPR, NICHD 



Others ; 



A. A. 
H.J. 



Herman 
Hoffman 



Visiting Scientist 
Chief 



EB, DESPR, NICHD 
EB, CD, NIDCD 



COOPERATING UNITS (if any) 

Norway National Institute of Public Health (L.S. Bakketeig) ; National Center for 

Health Statistics (C.J. Krulewitch) ; Chung Yuan Christian University (M.S. Yang) 



LAB/BRANCH 

Biometry and Mathematical Statistics Branch, DESPR 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
0.3 



PROFESSIONAL: 
0.3 



OTHER: 
0.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human 
D (al) Minors 
D (a2) Interviews 



n (b) Human 



E (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This Study will examine the intrinsic difficulty of estimating subpopulation 
characteristics when the identities of the observations are not completely known. 
Hard clustering and fuzzy clustering techniques to classify observations into 
subgroups are investigated. This study will develop estimation methodologies for 
the subpopulation characteristics. The statistical properties of the new 
methodologies will be studied. The new methodologies will be compared with the 
existing methodologies designed to remedy the misclassification problem. The new 
methodologies will alleviate some of the intrinsic difficulties for a heterogeneous 
mixture of subpopulations. Applications will be made to study the issues 
concerning biological heterogeneity and measurement error in the birth weight, 
gestational age and perinatal mortality. This study will also be applied to the 
investigation of intrauterine growth and pregnancy outcome. 



PHS 6040 (Rev. 5/92) 



1 



ZOl HD 00856-04 BB 

Pro.iect Description; 

Objectives: To develop statistical methods for studying subpopulation 
characteristics in a heterogeneous mixture of subpopulations. 

Methods Employed : Classical and modern statistical and probabilistic methods are 
employed. Computer technology is used as a tool for computation and simulation. 
New methodology is also developed. 

Ma.ior Findings : Estimation of subpopulation characteristics after a hard 
clustering of unidentified data into subpopulations is biased and inconsistent as 
long as there is a chance of misclassification. A notion of fuzzy partition of the 
data is introduced. It has been established analytically that the developed 
methodologies of estimation after fuzzy classification is unbiased under some 
general conditions. The variance of the new methodologies is also analytically 
established. Simulation studies have been conducted to compare the new 
methodologies and other existing methodologies and show that the new ones perform 
much better than the others in many measures. 

Significance to Biomedical Research and the Program of the Institute : Estimation 
after fuzzy clustering has been applied to estimate the perinatal mortality rates 
for the subpopulations of intrauterine growth retarded and the non-intrauterine 
growth retarded infants. It can also be used for other pregnancy outcomes. This 
methodology has also high potential for application in other substantive studies. 

Proposed Course of Pro.iect : Continuation of development of this methodology is 
proposed. Complete investigation of the analytic results as suggested by 
simulation studies is planned. Application to intrauterine growth and pregnancy 
outcome is to be investigated. Application to study birth weight distribution and 
gestational age is also planned. 

Publications: None 



b 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00857-04 BB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Methodological Research in Mathematical Statistics and Biostatistics 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.') (Name, title, laboratory, and institute affiliation) 
PI: Y.J. Lee Chief BMSB, DESPR, NICHD 



Others : J . Troendle 



Senior Staff Fellow 



BMSB, DESPR, NICHD 



COOPERATING UNITS fifanv) 

Hankook University of Foreign Studies (T. Park), Mayo Clinic (S. Jung), ATG (H. 

0" Grady) 



LAB/BRANCH 

Biometry and Mathematical Statistics Branch, DESPR 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
2.0 



PROFESSIONAL: 
2.0 



OTHER: 
0.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human 
D (al) Minors 
D (a2) Interviews 



n (b) Human 



C (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Methodological research in statistics is motivated often by interest in 
methodological/theoretical statistical questions. Consulting and collaborative 
projects also motivate the research because existing methods and theories may be 
inadequate to handle their data analysis or study design. 

Dr. Lee has completed a paper on a two-sample nonparametric test method for missing 
observations and presented as an invited paper at the August Multivariate 
Statistical Inference Conference. Dr. Lee has presented a number of invited 
statistical talks on clinical trial methodologies in South Korea and China. Drs. 
Lee and Park are working on a paper on incomplete data problems in clinical trials, 
and on a problem to improve coefficient estimates for repeated measure data. Dr. 
Troendle has been working on multivariate permutational problems as well as on 
combining multiple testing and global testing methods. Dr. Park has been working 
on multivariate regression methods. Drs. Lee and Jung have completed a paper on 
generalizing the logistic regression to be applied to survival data, and the paper 
has been accepted for publication in the Journal of the American Statistical 
Association , subject to revision. Drs. Lee and 0' Grady have been working on sample 
size problems for the two sample Wilcoxon test for ordinal categorical data. They 
are also working on nonparametric methods for the data from cross-over designs. 



PHS 6040 (Rev. 5/92) 



ZOl HD 00857-04 BB 



Pro.iect Description: 



Objectives : To develop statistical methods and theories for the analysis of 
complex biomedical data and for the design of new biomedical studies. 

Methods Employed : Both mathematical methods and computer simulation evaluation 
methods are applied to develop and evaluate new and existing statistical methods 
for design, implementation and evaluation of biomedical data analysis. 

Maior Findings : Dr. Lee has developed a nonparametric method for missing data and 
submitted a manuscript for publication. Drs. Lee and Park have completed 
simulation evaluations of statistical methods for two sample data where some are 
incomplete because of premature death of study subjects. Simulation evaluations 
show that the method assigning a worst possible score to the incomplete data is not 
statistically satisfactory. A paper has been submitted for publication. 

The logistic regression method is easy to understand and flexible to model the t- 
year survival probability. But when some data are censored, one cannot regress the 
t-year survival probability against covariates. Drs. Lee and Jung have proposed 
a maximum conditional likelihood approach to regressing the t-year survival 
probability. The method can be applied to estimating the survival distribution for 
given covariates. A paper has been submitted and accepted for publication in the 
Journal of American Statistical Association . 

Medical outcomes are often measured by ordered categorical scores such as severity 
score, performance status, etc. The two-sample discrete Wilcoxon test is broadly 
applied to analyzing the ordered categorical data. When designing studies with 
such outcome data, however, there is no satistisfactory method for determining the 
sample size. Drs. Lee and O'Grady have undertaken a methodological investigation 
for determining the sample size for the two sample discrete Wilcoxon test. 
Simulation evaluations show that the power of the test is minimized under the 
slipped configuration of probability distributions. They are now working on an 
analytical proof of their observation from the simulation evaluations. 

Dr. Troendle has been working on constructing a permutational step-up testing 
procedure for adjusting p values for multiple endpoints. Step-up tests are more 
powerful than step-down tests in certain situations. Although the algorithm 
derived for step-up adjusted p values is more computational than the existing step- 
down algorithm, some modifications have been made to reduce the computation. A 
paper has been submitted for publication. 

A simulation study of the properties of step-down permutation tests has been 
completed by Dr. Troendle and Clifford Blair. It was found that the step-down 
tests are particularly powerful when many endpoints are involved, the data are 
significantly correlated, or distributional assumptions are questionable. A paper 
has been accepted for publication. 

When multiple endpoints are involved, closed testing algorithms in conjunction with 
global tests have been proposed for use in identifying significant outcomes. An 
alternative is to use a stepwise assessment of the endpoints, where at each step 
an adjustment is made for the endpoint left. Dr. Troendle has completed a 
comparative study of some new and some existing methods for small sample sizes. 

Dr. Troendle has discovered the most powerful permutation test among all similar 
tests against a simple normal alternative in the multivariate two sample location 
shift problems. Unfortunately, no more powerful test exists for composite 






ZOl HD 00857-04 BB 

hypotheses. A simple approximation yields a new test which is applicable to 
composite one sided tests. The new test has been compared to Hotel ling's T^ 
permutation test and to Boyett and Shuster's maximal t test. A paper has been 
submitted for publication. 

Drs. Troendle, Yu and Herman have been working on standardization systems for birth 
weight and gestational age when the outcome measure is infant mortality. The 
analysis compares an ad hoc method of grouping infants into simple categories of 
birth weight (Wilcox, 1979) to a new procedure which extends the Wilcox method to 
incorporate gestational age while removing much of the ad hoc methods employed in 
that work. The method provides a decomposition of the infant mortality for a 
population into two sources: that expected for a standard population with a 
bivariate Gaussian distribution of birth weight-gestational age pairs centered at 
the population means and having the same birth weight-gestational age specific 
mortality, and the additional mortality obtained because of the actual skewed 
distribution of births. Applied to U.S. data, the new method shows a contrast to 
the conclusions of Wilcox and Russell (1986). 

Dr. Park proposed a general class of multivariate regression models which can 
handle both discrete and continuous repeated measurements. The proposed model is 
based on the seemingly unrelated regression models. The regression parameters of 
the model care was consistently estimated using the two-stage least squares method. 
A paper has been published. 

Drs. Park and Brown consider categorical data with binary responses subject to non- 
ignorable non-responses. When fitting log-linear models for these data, they 
showed that the ML estimation method may often yield estimators with infinity or - 
infinity. They proposed a constrained ML estimation by restricting the parameter 
space of models. With a reasonable choice of boundary constraints, they show that 
the proposed estimators perform better than the ML estimators. A paper has been 
submitted for publication. 

The generalizing estimating equations approach is widely applied to analyze 
repeated categorical data. In this approach, the correlations among the 
observations from the same subject has been estimated using the Pearson residuals. 
For discrete distributions, these Pearson residuals are not normally distributed. 
Drs. Park, Davis and Li have considered using different residuals: Anscombe and 
deviance which are more normally distributed than the Pearson residuals. They 
compared three residuals using the simulation studies. The results have been 
summarized into a paper which has been submitted for publication. 

Drs. Lee and Park continue to work on obtaining well-defined residuals which are 
more normally distributed than Pearson, Anscombe or Deviance residuals. Drs. Lee 
and Park have considered Box-Cox types of transformations and have undertaken 
simulation studies to investigate their properties. 

In repeated measures studies, the main interest often lies in comparing groups 
effects. The comparison of group effects can usually be performed by testing the 
equality of group means. Sometimes groups are formulated in an order relation. 
Dr. Park proposed a test procedure for testing the equality of group means in this 
situation and showed that the proposed method is more powerful than the 
conventional one. A paper has been submitted for publication. 

Significance to Biomedical Research and the Program of the Institute : We will be 
better able to analyze biomedical data and design studies. 



b 



ZOl HD 00857-04 BB 

Proposed Course of Pro.iect : Continue to explore and develop statistical methods 
applicable to biomedical data and projects. 

Publications : 

Troendle J. A stepwise resampling method of multiple hypothesis testing. J Am 
Stat Assoc 1995;90:370-8. 

Troendle J, Yu K. A note on testing the Hardy-Wei nberg Law across strata. Ann of 
Hum Gen 1994;58:397-402. 

Jung SH, Lee YJ. Logistic regression of t-year survival probability: Maximum 
conditional likelihood method. J Am Stat Assoc, in press. 

Blair RC, Troendle JF, Beck RW. Control of family wise errors in multiple endpoint 
assessments via stepwise permutation tests. Stat Med, in press. 

Park T. Multivariate regression models for discrete and continuous repeated 
measurements. Com Stat Theory Meth, in press. 

Park T, Brown MB. Methods for categorical data with nonignorable nonresponse. J 
Am Stat Assoc 1994;89:44-52. 



u 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NXJMBER 

ZOl HD 00859-04 BB 



PERIOD COVERED 

October 1, 1994 to September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 
Meta-Analytic Methods 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.') (Name, title, laboratory, and institute affiliation) 
PI: Rebecca DerSimonian Mathematical Statistician BMSB, DESPR, NICHD 



COOPERATING UNITS fifanv) 



LAB/BRANCH 

Biometry and Mathematical Statistics Branch, DESPR 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
0.60 



PROFESSIONAL: 
0.60 



OTHER: 
0.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human D (b) Human O (c) Neither 

D (al) Minors 

D (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project consists of four components which continue from last year: The first 
compares a fixed and a random effects model for combining data from a series of 
clinical trials. The study evaluates the performance of each method under various 
heterogeneity assumptions and with several scales of measurement, including the 
risk difference and the odds ratio as measures of effect. 

The second develops and compares parametric and non-parametric tests to assess the 
assumption of homogeneity of effects in data from a series of trials. This 
component considers data from Gaussian, binomial, and Poisson sampling 
distributions . 

The third considers meta-analytic methods for combining the evidence from a series 
of HIV seroprevalence studies to estimate HIV prevalence in a target population. 
Sentinel studies are typically investigations of incompletely defined cohorts which 
are convenient to survey but are often based on non-probability samples. Self- 
selection and similar issues inherent in sentinel studies makes generalizing 
results from a single study to a target population problematic. This research 
assesses the use of formal meta-analytic methods for HIV prevalence estimation in a 
target population by incorporating information from several HIV sentinel 
seroprevalence studies. Preliminary results from this component is to appear in 
Annals of Epidemiology (in press). 

The fourth component addresses issues that pertain to the use of meta-analysis in 
t;he design and monitoring of clinical trials. This research evaluates the role of 
formal incorporation of external evidence summarized from a meta-analysis of 
previous or concurrent results into sample size considerations and stopping rules 
during the conduct of a clinical trial. Preliminary results from this component is 
to appear in Statistics in Medicine. 



PHS 6040 (Rev. 5/92) 



lO 



ZOl HD 00859-04 BB 



Pro.iect Description: 



Objectives: To develop statistical methods for combining data from a series of 
clinical trials and HIV seroprevalence sentinel studies. 

Methods Employed : For meta-analysis of clinical trials, this project considers 
fixed and random effects as well as Bayes models for meta-analysis of data under 
various heterogeneity assumptions and several scales of measurement, including the 
risk difference and the odds ratio as measures of effect. Both parametric and non- 
parametric methods are employed for assessing homogeneity of effects. The role of 
meta-analysis in the design and monitoring of a new clinical trial is also 
considered. 

For meta-analysis of HIV seroprevalence studies to estimate prevalence in the 
target population, this project considers empirical Bayes methods to incorporate 
information from related sentinel studies. This research characterizes the special 
epidemiologic and statistical issues inherent in sentinel studies for HIV 
prevalence estimation and develops methodology that addresses them when 
incorporating the information from several such studies. 

Major Findings : For meta-analysis of clinical trials, several tests of 
heterogeneity for binomial, Gaussian, and Poisson sampling distribution are 
developed and compared. Fixed and random effects, as well as Bayes models, are 
evaluated and compared under various heterogeneity assumptions. Furthermore, the 
impact of using meta-analytical results in the design and monitoring of the NICHD 
trial on preeclampsia is evaluated (to appear in Statistics in Medicine ). 

For meta-analysis of seroprevalence studies, we find that prevalence in an 
individual HIV sentinel serosurvey is time-averaged and vulnerable to several time- 
dependent biases and that self-selection and laboratory methods are additional 
likely sources of bias. Incorporating the information from several such HIV 
sentinel seroprevalence studies mitigates the possible impact of these biases 
inherent in the individual studies (to appear in Annals of Epidemiology ). 

Significance to Biomedical Research and the Program of the Institute : The 
statistical methods developed in this project are applicable to many data sets 
relevant to the Institute where the evidence on a particular topic is conflicting 
and/or the sample sizes in the available studies are too small to yield unequivocal 
results. An example of the applicability of the methods to relevant problems in 
the Institute is the workshop organized by the Division on "The Role of Meta- 
Analysis in the Design and Monitoring of Clinical Trials." Being directly relevant 
to this topic, preliminary results from this project were presented at the 
workshop, which was prompted by concerns raised at the Data Safety and Monitoring 
Board of the large multi-center trial conducted by the Institute on "Calcium 
Supplementation for the Prevention of Preeclampsia." 

Proposed Course of Project : Continue to explore and develop the methodology for 
tests of homogeneity, particularly the non-parametric ones, to further evaluate the 
role of meta-analysis in planning a new clinical trial, and to develop methods for 
combining data from HIV sentinel studies, especially when covariate information may 
be missing in some studies. 

Publications : 

Strickler H, Hoover DR, DerSimonian R. Problems in interpreting HIV sentinal 
seroprevalence studies. Ann Epidemiol 1995. (In Press) 

i: 



ZOl HD 00859-04 BB 

DerSimonian R. Meta-analysis in the design and monitoring of clinical trials 
Stat Med 1996. (In Press) 



1 






DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 01802-05 CSB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Data Coordinating Center for the NICHD Study of Early Child Care 



PRINCIPAL INVESTIGATOR fl^ist other professional personnel below the Principal Investigator.') (Name, title, laboratory, and institute affiliation) 
PI: B.K. Knoke Computer Specialist (10/94-5/95) CSB, DESPR, NICHD 

A.C. Trumble Computer Specialist (6/95-9/95) CSB, DESPR, NICHD 



COOPERATING UNITS fifanv) 
CRMC, NICHD/NIH 



LAB/BRANCH 

Computer Sciences Branch, DESPR 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, MD 20892 



TOTAL STAFF YEARS: 
1.6 



PROFESSIONAL: 
.85 



OTHER: 

.75 



CHECK APPROPRIATE BOX(ES) 

B (a) Human 
ill (al) Minors 
£] (a2) Interviews 



n (b) Human 



n (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This Study is a 10-site cooperative agreement to examine the effects of non- 
maternal care, especially during the first year of life. It focuses on the social 
and intellectual development of children in an ecological framework which takes 
into account the complex interactions of non-maternal care experiences with home 
and family conditions, parenting practices, and child characteristics. Over 1200 
families were enrolled in the study during 1991. These families will participate 
in the study for 36 months. 

The Data Coordinating Center (DCC) is responsible for data management and protocol 
monitoring. It serves as the focal point for receipt of collected data and for 
data processing. 

The DCC is currently processing 24 and 36 month data. The data are entered into 
computer files , edited and summarized for monitoring purposes . Routine edit 
reports are being distributed to the sites regularly. Project monitoring reports 
are distributed to the Steering Committee quarterly. 



PHS 6040 (Rev. 5/92) 






ZOl HD 01802-05 CSB 

Pro.iect Description 

Objectives: To examine the effects of non-maternal care, especially during the 
first year of life on the social and intellectual development of young children. 
The Data Coordinating Center is located in the Computer Sciences Branch of the 
Division of Epidemiology, Statistics and Prevention Research. The objective of 
the Data Coordinating Center is to assure that the protocol is uniformly 
administered at all sites. This includes the preparation of Manuals of 
Operations; management of monitoring subject recruitment; design of data 
collection instruments, data entry methods and data base management systems; and 
generation of statistical reports. 

Methods Employed : At each of the ten sites participating in the study, 
approximately 120 infants and their parents were enrolled in this study shortly 
after the infant's birth and will be followed until the infant is 3 years old. 
Over the 36 months of participation, the infant will be visited and observed in 
the home, in child care, and in a laboratory setting. The sample was selected 
using a weighted method with the aim of recruiting a minimum of 10% minority, 10% 
low education and 10% single parent families. In addition, the sample was 
weighted to contain 60% mothers planning to return to work full time, 20% 
planning to work part-time and 20% not planning to return to work. 

Through close monitoring of the sample recruitment, a sample has been selected 
which closely represents the goal. This study is being conducted in 
collaboration with the Center for Research for Mothers and Children. 

Major Findings : All findings are preliminary at this time. 

Significance to Biomedical Research and the Program of the Institute : This 
project will help determine the extent and type of non-maternal care used in this 
country and the effects that different patterns of care have on the development 
of children in the first three years of life. 

Proposed Course : The data collection for phase I is almost complete. The Data 
Coordinating Center for phase II will be carried out under a grant. 

Publications : 

Friedman S, The NICHD Early Child Care Network. Child care and child 
development: The NICHD study of early child care. In: Friedman S, Haywood HC, 
eds. Developmental follow-up: concepts, domains and methods. San Diego: 
Academic Press, 1994;337-96. 






DIVISION OF EPIDEMIOLOGY, STATISTICS AND PREVENTION RESEARCH 
Computer Sciences Branch 

Bijur PE, Trumble AC, Harel Y, Overpeck MD, Jones HD, Scheldt PC. Sports and 
recreation injuries in U.S. children and youth. Arch Pediatr Adolesc Med, in 
press. 

Friedman S, The NICHD Early Child Care Network. Child care and child 
development: The NICHD study of early child care. In: Friedman S, Haywood 
HC, eds. Developmental follow up: concepts, domains and methods. San Diego: 
Academic Press, 1994;337-95. 

Harel Y, Overpeck MD, Jones, Scheidt PC, Bijur PE, Trumble AC. The quality of 
proxy-respondent data in NCHS Surveys. [Letter to Editor] Am J Public Health 
1995;85:591. 

Overpeck MD, Trumble AC, Brenner R. Population-based surveys as sources of 
U.S. injury data and special methodological problems. Proceedings of the 
International Collaborative Effort on Injury. Hyattsville, MD: National 
Center for Health Statistics, 1995; DHHS publication no. (PHS)95-1252;12-17. 

Scheidt PC, Harel Y, Trumble AC, Jones DH, Overpeck MD, Bijur PE. 
Epidemiology of non-fatal injuries in children and youth. Am J Public Health 
1995;85:932-38. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 01703-06 ESPR 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Study of Pregnancy Outcome, Maternal Death and Child Health in Pakistan 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.') (Name, title, laboratory, and institute affiliatiort 

PI: H.W. Berendes Director DESPR, NICHD 



COOPERATING UNITS fifanv) 

Department of Community Health, Aga Khan University (Drs . Joseph McCormick, Fariyal 
Fikree, Farid Midhet, and Mehtab Karim) , Special Program for Research, Reproduction 
and Family Planning of the WHO fPr. Jose Villar') 



LAB/BRANCH 

Office of the Director, DESPR 



SECTION 



INSTITUTE AND LOCATION 



NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
0.2 



PROFESSIONAL: 
0.1 



OTHER: 
0.1 



CHECK APPROPRIATE BOX(ES) 

H (a) Human 
C (al) Minors 
£3 (a2) Interviews 



D (b) Human 



n (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 
This project has three components. 

The Maternal and Infant Mortality Survey (MIMS) will provide estimates of maternal 
and infant mortality rates from population based samples in Karachi and the four 
provinces of Pakistan. In addition, the Survey will provide information on causes 
of maternal and infant deaths and demographic and other determinants of maternal 
and infant mortality. 

The Pregnancy Outcome Study (POS) is a cross sectional and prospective study of 
pregnant women from selected katchi abadies to identify determinants of poor birth 
outcome in this population. 

The Child Survival Study (CSS) is a follow up study of live born children from POS 
through two years of age to assess mortality, morbidity and physical and 
developmental measures . 

A new component is the development of an intervention to reduce infant mortality 
and morbidity by training all levels of care providers (dais, lady health visitors, 
midwives , physicians) to recognize timely high risk conditions during pregnancy or 
at delivery, set up a referral and transportation system and get high risk women 
timely referral to a tertiary medical facility. 



PHS 6040 (Rev. 5/92) 



ZOl HD 01703-06 ESPR 

Pro.iect Description 

Objectives: Based upon the findings on the Maternal and Infant Mortality Surveys 
an intervention is being developed in Karachi to reduce maternal and infant 
mortality. Specifically what is proposed is to conduct a study to evaluate the 
effectiveness of an intervention strategy which will consist of training, 
monitoring and supervision of providers, setting up a referral and transport 
system to tertiary care providers of high risk women and women with complications 
around the time of labor and delivery and information, education and 
communication campaign to increase awareness at the community level. 

Methods Employed : The Maternal and Infant Mortality Survey (MIMS) is a two step 
survey. The first step consists of an enumeration of household composition over 
the last five years and identification of any deaths occurring during that 
period. The second step is a detailed interview (verbal autopsy) of household 
members to identify pregnancy related deaths, to obtain information about 
possible cause of maternal deaths, as well as of infant deaths. The survey in 
Karachi will be based upon sites which are currently part of a primary health 
care network developed by the Department of Community Health of the Aga Khan 
University and will include approximately 10,000 households. There will 
additionally be surveys done in each of the four provinces, that is, Baluchistan, 
Sindh, Northwest Frontier Territory, and the Punjab. Sample sizes for the 
provincial surveys will vary between 10-20,000 households depending on 
availability of funds. Cluster samples will be taken from select sites of each 
province. 

PCS will identify all pregnant women at a given time in the katchi abadies which 
are part of the primary health care system of the Aga Khan University Department 
of Community Health and follow them to delivery to identify determinants of poor 
birth outcome. Information is collected through questionnaire and by physical 
examination. Since most women deliver at home attended by traditional birth 
attendants, a notification system is in place notifying the project about any 
births within two days to obtain birth weight as well as modified Dubowitz 
evaluation to differentiate preterm births from intrauterine growth retardation 
among low birth weight children. The Pregnancy Outcome Study is completed. 

CSS is a prospective follow up of live births from POS to age two to assess 
mortality and morbidity and determinants of physical growth and development. The 
follow up will include interviews, physical examination, measurements of physical 
growth, as well as behavioral assessments. The Child Survival Study is 
completed. 

It is proposed to implement an intervention in a section of Karachi called 
Korangi consisting of ten contiguous wards with a population of approximately 
500,000. As controls, another site is chosen nearby of similar size and similar 
population characteristics. Both intervention and control sites will be 
subjected to a pre- and post-intervention survey to obtain information on current 
maternal and infant mortality rates and complications around the time of 
pregnancy. 



^ 



ZOl HD 01703-06 ESPR 



Major Findings : The MIMS Surveys have been completed now in Karachi, four sites 
in Baluchistan and two sites in the Northwest Frontier Province which in total 
include approximately 36,000 households. While the overall maternal mortality 
rate is high in all sites, there is considerable variation between sites, the 
lowest rate is in Karachi and was 2.7/1000 live births and the highest in parts 
of Baluchistan of about 12/1000. Infant mortality rates vary accordingly, also 
from a low of about 75/1000 to a high of 210/1000 live births. This range 
represents different katchi abadies within the city of Karachi. During the past 
year, surveys in the Northwest Territory have been completed and analyses are in 
progress. A new survey of MIMS in Sindh Province has been initiated. 

Considerable progress has been made in the analysis of data from the Pregnancy 
Outcome Study. Current analyses deal with risk factors related to intrauterine 
growth retardation which is a major risk factor in developing countries and 
accounts for the major fraction of low birth weight births in developing 
countries. Analysis of the data suggests that major risk factors include 
socioeconomic conditions, including housing construction, paternal and maternal 
education, paternal employment and the source of water supply, as well as ethnic 
differences apparent among the different ethnic groups in these communities which 
consists of Hindus, Muslims and Christians. Biological factors related to the 
risk of lUGR include young maternal age, primiparity and high parity, history of 
poor prior pregnancy outcome, a short interconception interval, low maternal 
height and weight, as well as a low mid-arm circumference and low skinfold 
thickness reflecting maternal nutritional status as well as a non-vegetarian diet 
during pregnancy. In addition, there was a marked increase in risk due to 
consanguinity. 

In a multi-variate analysis, remaining risk factors included the source of water 
supply, maternal education and paternal employment, primiparity and grand 
multiparity, consanguinity, less than one year interval between birth to 
conception, low maternal height and weight and also non-vegetarian diet. These 
findings suggest possible intervention especially related to improved nutrition 
but also raising the status of women by mandating primary education of all school 
age girls. 

The intervention is currently under development and will begin in 1996. 

Significance to Biomedical Research and the Program of the Institute : The 
identification of determinants of maternal and infant mortality is a major 
research interest of the Institute and the findings of this project have specific 
public health importance to the country of Pakistan and other countries who are 
in a similar stage of development. A conference was held in the spring of 1994 
to present the findings from the Maternal and Infant Mortality Surveys to an 
audience consisting of representatives of Ministries of Health, the Provincial 
Government, leading academicians and representatives of international health 
agencies for the purpose of acquainting this audience to the findings from the 
surveys but also as a means of developing possible interventions to address the 
high maternal and infant mortality. The World Bank participated in the planning 
for this conference and underwrote some of the costs. The proceedings of the 
conference are in preparation for publication. 



3 



ZOl HD 01703-06 ESPR 

The interventions to reduce maternal and infant mortality in developing countries 
is clearly of major public health significance and of interest to this Institute. 

Proposed Course : The intervention is currently under development with active 
participation of the Director of the Division. Funding is provided by the World 
Bank and by AID with some funding expected also from UNICEF in Pakistan. The 
Director, DESPR will serve as a senior technical advisor in this project with 
active participation in its design and implementation as well as the analysis of 
the data. 

Publications : 

Fikree F, Berendes H. Risk factors for term intrauterine growth retardation: A 
community-based study in Karachi. Bull WHO 1994;72(4) :581-7. 

Fikree FF, Gray RH, Berendes HW, Karim MS. A community-based nested case-control 
study of maternal mortality. Int J Gyn Obstet 1994;47:247-55. 

Fikree FF, Berendes HW, Villar J. A rapid nutritional evaluation of pregnant 
women in low socioeconomic settlements of Karachi, Pakistan. J Pakistan Med 
Assoc, in press. 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 01704-05 ESPR 



PERIOD COVERED 



October 1, 1994 through October 31, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 
Stunting Among Bedouin Arab Children in the Negev, Israel 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 
PI: H.W. Berendes Director DESPR, NICHD 



COOPERATING UNITS fifanv) 

Cancer Prevention Studies Branch, DCPC, NCI, NIH (M.R. Forman) ; Ben Gurion 

University of the Negev, Beer Sheva, Israel (G. Hundt) 



LAB/BRANCH 

Office of the Director, DESPR 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



_2_ 



L2_ 



TOTAL STAFF YEARS: 
0.05 



PROFESSIONAL: 
0.05 



OTHER: 
0.0 



CHECK APPROPRIATE BOX(ES) 

B (a) Human 
£] (al) Minois 
C (a2) Interviews 



D (b) Human 



n (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 
This project has been completed. 



PHS 6(H0 (Rev. 5/92) 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 01705-02 ESPR 



PERIOD COVERED 

October 1, 1994 through December 1, 1994 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 
Women's Lifestyles in Pregnancy Study, Analysis of Data 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.') (Name, title, laboratory, and institute affiliation) 
P.I.: H. W. Berendes Director DESPR, NICHD 



COOPERATING UNITS fifanv) 

The David and Lucile Packard Foundation (Dr. Patricia H. Shiono) ; Center for 

Population, Columbia University (Dr. Virginia A. Rauh) 



LAB/BRANCH 

Office of the Director, DESPR 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
0.05 



PROFESSIONAL: 
0.05 



OTHER: 
0.0 



CHECK APPROPRIATE BOX(ES) 

B (a) Human 

n (al) Minors 

£] (a2) Interviews 



D (b) Human 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The analysis of data has been completed. 



PHS 6040 (Rev. 5/92) 



b 



DEPAKIMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT mJMBER 



ZOl HD 01706-01 ESPR 



PERIOD COVERED 

November 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

A Randomized Controlled Trial for the Evaluation of a New Antenatal Care Model 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.') (Name, title, laboratory, and institute affiliation) 
P.I.: H.W. Berendes Director DESPR, NICHD, NTH 



COOPERATING UNITS fifanv) 

WHO, Geneva (J. Villar) ; Rosario Centre for Perinatal Research, Rosario, Argentina 
(J.M. Belizan) ; Sites in Saudi Arabia (R. Al-Mazrou) ; Havana (U. Far not) ; South 
Africa (J. Moodl eyV Thailand (¥ . Lumbiganon') 



LAB/BRANCH 

Office of the Director, DESPR 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
0.2 



PROFESSIONAL: 
0.2 



OTHER: 
0.0 



CHECK APPROPRIATE BOX(ES) 

Q (a) Human 
G (al) Minors 
£} (a2) Interviews 



n (b) Human 



n (c) Neither 



SUMMARY OF WORK (Use standard uru'educed type. Do not exceed the space provided.) 

This project consists of a randomized controlled clinical trial comparing two 
models of antenatal care. As background, a few years ago, the Institute sponsored 
with other agencies of the Public Health Service an Expert Panel on Prenatal Care 
which indicated that the current system of care, either in the schedule of visits 
as well as its contents is for the most part not based upon a careful scientific 
assessment. The recommendation of the Panel was to revise the schedule of prenatal 
care for low risk women in order to save resources which can be used for high risk 
women. It was also recommended that studies or trials be developed to evaluate 
different models of prenatal care. Prenatal care is viewed as a major determinant 
of infant mortality and morbidity and its careful evaluation obviously is of major 
research interest to the Institute. 

The WHO through a technical advisory panel has developed over the last two years a 
new model for prenatal care for developing countries which reduces the number of 
visits and also alters the content of prenatal care substantially. In the revised 
model, the content of prenatal care will consist predominately of the recognition 
and diagnosis of conditions and complications of pregnancy which are known to 
affect the health of women or birth outcome and specific inter-ventions to deal with 
these conditions once identified. The DESPR, through its Director, has been an 
integral part of the design of this new model of prenatal care and has served as 
the Chair of the Technical Advisory Panel of the WHO. 

It is proposed that this trial be evaluated in four or five sites by randomizing 
clinics in these four or five sites. Clinics would be randomized to the current 
system of prenatal care or the new proposed model of prenatal care . There would be 
at least 12 clinics per participating center for a total of 24 clinics receiving 
the new model of prenatal care and 24 with the current system. 



PHS 6040 (Rev. 5/92) 



ZOl HD 01706-01 ESPR 

Project Description 

Objectives: To conduct a randomized clinical trial comparing two models of 
antenatal care to establish the relative merits of each model. It is intended to 
test whether the proposed new model is as effective as the traditional multi-visit 
model with regard to maternal mortality and perinatal morbidity and mortality, and 
satisfaction. 

Methods Employed ; This study will be conducted in four or five sites. The sites 
consist of Rosario, Argentina; Havana, Cuba; South Africa; Thailand; and Saudi 
Arabia. Randomization will be by care provider units or clinics rather than by 
patients with provider units or clinics being assigned to the current regime of 
prenatal care or the new model to be tested. There will be a total of 48 units or 
clinic sites being randomized and the patient population enrolled in this trial 
will be somewhere between 24-28,000. 

Ma.ior Findings : None 

Significance to Biomedical Research and the Program of the Institute : To test new 
models of prenatal care is of major research interest and public health importance 
in the field of maternal and child health. Clearly if it could be shown that a 
revised model of prenatal care consisting of fewer visits and focusing on 
conditions of pregnancy known to be affecting outcome is 

equivalent in outcome to what is currently in place, this would result in a major 
saving of resources which could then be used for dealing with high risk 
pregnancies. 

Proposed Course : The protocol for this project has been developed and the sites 
have been identified. The study is expected to start between October 1, 1995 and 
January 1, 1996 in the four or five sites. The trial will run approximately two 
years and the results should be available in fiscal year 1998. 

The Division supports one site in Rosario through a contract with funding for the 
other sites provided by different donor agencies and WHO. 

Publications: None 



o 



DEPAFTTMEhn" OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SERVICE 

NOTICE OF im"RAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00331-12 EB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Diabetes In Early Pregnancy (DIEP) 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.} (Name, title, laboratory, and institute affiliation) 

PI: J.L. Mills Chief, Pediatric Epidemiology Section EB , DESPR, NICHD 



Other: M.R. Conley 
Y.J. Lee 



Computer Specialist 
Chief 



EB, DESPR, NICHD 
BMSB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Cornell Univ. Med. Center , NY (L. Jovanovic) ; Brigham and Womens Hosp. , Boston, MA 
(L.Holmes); Northwestern Univ. Med. Center , Chicago, IL (J.L.Simpson); Univ. of 
Pittsburgh. Pittsburgh. PA (J.Aarons): Univ. of Washington. Seattle. WA (R.Knopp) 



LAB/BRANCH 

Epidemiology Branch 



SECTION 

Pediatric Epidemiology Section 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 




PROFESSIONAL: 






OTHER 


1.0 




1.0 









CHECK APPROPRIATE BOX(ES) 










(a) Human subjects 


n 


(b) Human tissues 


n 


(c) Neither 


B (a1) Minors 










EI (a2) Interviews 













SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The Diabetes in Early Pregnancy Project was designed 1) to examine the relationship 
between maternal diabetic control during organogenesis and malformations in the 
offspring, and to identify, if possible, a specific teratogenic factor or factors 
in the diabetic metabolic state; and 2) to compare early fetal loss rates in women 
with diabetes and in non-diabetic control subjects. ¥e found that diabetic women 
who came into care before the period of organogenesis achieved better results than 
those who came in later; but their results were still poorer than for non-diabetic 
control subjects. Differences in maternal glucose levels during organogenesis did 
not explain the malformations in the offspring of the women who were followed 
throughout pregnancy. These results suggest that women who enter late (and were 
not under medical supervision during organogenesis) probably had poor control. 
This resulted in malformations due to hyperglycemia or related factors. The 
results from the diabetic group entering early strongly suggest that other 
teratogenic mechanisms were present. Regarding early fetal losses, we found that 
diabetic women in good metabolic control were at no higher risk for spontaneous 
abortion than control women; the risk of loss increased dramatically as diabetic 
control worsened; and the overall risk of losing a pregnancy was lower than 
expected, only 16Z. Since these primary analyses were completed, a number of 
related studies have been completed (see previous reports). An analysis of the 
effect of metabolic control on the progression of retinopathy was published this 
year. Values of glycosylated protein and fructosamine have been tised to study the 
effect of intermediate first trimester control on fetal loss. This analysis is 
being written up for publication. A study of infection as a risk factor for early 
spontaneous abortion has been completed and is about to be submitted. Assays of 
anti-sperm, anti-phospholipid , and anti-cardiolipid antibodies have been completed 
and their relationship to fetal loss is being studied. Change in insulin 
requirement during pregnancy is being analyzed. 



PHS 6040 (Rev. 5/92) 



ZOl HD 00331-12 EB 

Pro.iect Description : Dr. James L. Mills, Dr. Jack Lee and Ms. Mary Conley of the 
DESPR are involved in data editing and analysis. The principal investigators 
noted on form 6040 also take an active part in data analysis and reporting. 

Objectives : The objectives of the DIEP were to examine the relationship between 
maternal diabetic control during organogenesis and malformations in the 
offspring; to identify, if possible, a specific teratogenic factor in diabetic 
metabolic state; and to compare early fetal loss rates in women with diabetes and 
non-diabetic control subjects. Secondary goals include investigating the 
metabolic milieu in early pregnancy, determining risk factors for spontaneous 
abortion in the non-diabetic population, and relating metabolic events in 
diabetic pregnancy to complications such as retinopathy in the mother and minor 
malformations in her offspring. A number of issues relating to metabolic events 
in diabetic pregnancy, malformations and retinopathy are now being examined. 

Methods Employed : An innovative study design in which women were identified 
prior to becoming pregnant and monitored closely for early diagnosis of pregnancy 
was used to address these questions. Women were requested to enroll prior to 
becoming pregnant and in over 50% of cases subjects monitored basal body 
temperature in order to identify pregnancy at the earliest possible time. Data 
were gathered on all pregnancy losses. Malformations were identified at delivery 
or, when feasible, after abortion. Data from these pregnancies are now being 
analyzed. Repository specimens collected in anticipation of future need are now 
being used to answer a variety of questions. 

Major Findings : The major questions have been answered. We found that diabetic 
women who came into care before the period of organogenesis achieved better 
results than those who came in later; but their results were still poorer than 
for non-diabetic control subjects. Differences in maternal glucose levels during 
organogenesis did not explain the malformations in the offspring of the women who 
were followed throughout pregnancy. These results suggest that women who enter 
late (and were not under medical supervision during organogenesis) probably had 
poor control. This resulted in malformations due to hyperglycemia or related 
factors. Data from the diabetic group entering early strongly suggest that other 
teratogenic mechanisms were present. Regarding early fetal losses, we found that 
diabetic women in good metabolic control were at no higher risk for spontaneous 
abortion than control women; the risk of loss increased dramatically as diabetic 
control worsened; and the overall risk of losing a pregnancy was lower than 
expected, only 16%. 

This past fiscal year we published the results of our analysis of the 
relationship between metabolic control and retinopathy. First trimester 
infection does not appear to be a risk factor for early spontaneous abortion. 
Autoimmune phenomena which have been implicated in second trimester loss do not 
appear to be significant factors in earlier loss. 



(v 



ZOl HD 00331-12 EB 

Significance to Biomedical Research and the Program of the Institute : Our major 
findings have been that poorly controlled diabetes increases the risk of 
spontaneous abortion. The risk can be eliminated by good control. Congenital 
malformations can be reduced by good periconceptional care. However, factors 
other than glucose are teratogenic and elevated malformation risks are still 
present in women in moderately good metabolic control. 

Our findings on progression of diabetic retinopathy during pregnancy have 
important clinical implications. Diabetic women who have moderate or worse 
retinopathy at the beginning of pregnancy are at high risk for progression and 
possible loss of vision. Such women must be monitored closely and treated 
promptly for signs of proliferate retinopathy. In order to reduce the risk of 
progression, women must be in very good control prior to conception. It was not 
known how good control must be to reduce the risk of complications. Our access 
to pre-loss blood samples which is unique is enabling us to investigate a variety 
of postulated risk factors not previously studied in very early pregnancy. 

Proposed Course of Pro.iect ; The DIEP is completing an analysis of glycosylated 
proteins and malformation outcomes. We will continue to look at the relationship 
between ketone levels and bad pregnancy outcomes, and we will examine potential 
patterns of minor malformations in infants with diabetic mothers. Anti- 
phospholipid antibodies as a risk factor for spontaneous abortion will be 
studied. A postulated drop in insulin requirement during the first trimester is 
being investigated. 

Publications: 



DEPARTMENT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SERNflCE 

NOTICE OF INTTRAMURAL RESEAFtCH PROJECT 



PROJECT NUMBER 



ZOl HD 0334-12 EB 



PERIOD COVERED 



October 1, 199; through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Low Birth Weight Across Generations 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 



PI: M.A. Klebanoff 



Research Medical Officer 



EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Office of the Director, DESPR, NICHD (H.W.Berendes ) ; University of Pennsylvania 
(S.Katz), University of Southern California (B.Mednick) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD. NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

.25 



PROFESSIONAL 



,25 



CHECK APPROPRIATE BOX(ES) 

H (a) Human subjects 
D (a1) Minors 
Q (a2) Interviews 



n (b) Hunnan tissues 



n (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The original description of the correlation between birthweights of mothers and 
their infants was followed by the description of the association between large 
maternal birth weight and delivery of a macrosomic (>4000 gram) infant. Study of 
other fetal growth parameters, including length and head circumference, 
demonstrated that infants of low birth weight mothers were both shorter and lighter 
than infants of larger mothers, but that the infants were normally proportioned. 

In ensuing studies, birth certificates of infants born in Tennessee between 1979 to 
1984 were matched with those of their mothers, who were born in Tennessee between 
1959 to 1966. Maternal and infant birth weights were again shown to be correlated. 
In addition, women who were themselves of low birth weight were up to 4 times as 
likely to have a small-f or-gestational age infant as were women who weighed 4000- 
4499 grams at birth, but low birth weight women were less than twice as likely to 
have a preterm infant. A group of Swedish women, born from 1955 to 1965, was next 
studied. Women who themselves were small-f or gestational age at birth were at 
increased risk of giving birth to both small-f or-gestational age and preterm 
infants. Women who were preterm at birth were not at increased risk of either 
outcome . 

Analysis of data from girls who were born in the 1960 's as subjects in the 
Collaborative Perinatal Project and Danish Perinatal Study is currently underway in 
order to examine their reproductive histories. Small-f or-gestational age, preterm 
and control girls have been located and interviewed. Hospital records of their 
deliveries have also been retrieved. 



PHS 6040 (Rev. 5/92) 



ZOl HD 00334-12 EB 
Pro.ject Description 
Ob.iectives ; 

1) Evaluation of the association between the birth weight of a mother with those 
of her children. 

2) Determination of whether the relationship is due to the presence of outliers, 
or due to a shifting of the entire birth weight distribution in the second 
generation. 

3) Separation of the second-generation effect of maternal birth weight into 
independent effects of maternal gestational age at delivery and maternal 
intrauterine growth. 

4) Study of the effects of the perinatal experience of mothers on their 
subsequent reproductive and health-related outcomes. 

Methods Employed ; Work done prior to FY94 has addressed objectives 1 and 2. 
Work continuing in FY94 is directed at addressing objectives 3 and 4. Two 
studies of similar design are currently being analyzed. In both of these 
studies, reproductive-age women whose own births had been extensively documented 
due to their participation in a study of pregnancy were located, and their 
pregnancy outcomes determined. One study, done under contract by the University 
of Pennsylvania (subcontracting to Brown University), has traced approximately 
120 women who were themselves born preterm, 160 who were small-for-gestational 
age (SGA), and 350 term, appropriately grown controls. These women were 
originally subjects in the Philadelphia and Providence cohorts of the 
Collaborative Perinatal Project. Reproductive histories have been obtained from 
these women, and medical records from their deliveries have been abstracted. A 
second contract, awarded to the University of Southern California (subcontracting 
to the Psykologisk Institut in Copenhagen), has traced approximately 158 parous 
women who were themselves preterm, 150 who were SGA, 37 who were both preterm and 
SGA, and 943 parous term controls. These women were originally subjects in the 
Danish Perinatal Study (1959-61). The extensive record linkage system in Denmark 
has enabled the collection of data from nearly 100% of all second-generation 
pregnancies. In addition, the Danish record linkage system has enabled the 
collection of data on paternal birth weight and (through military draft records) 
paternal adult height and weight. 

Major Findings ; The original description of the correlation between birthweights 
of mothers and their infants was followed by the description of the association 
between large maternal birth weight and delivery of a macrosomic (>4000 gram) 
infant. Study of other fetal growth parameters, including length and head 
circumference, demonstrated that infants of low birth weight mothers were both 
shorter and lighter than infants of larger mothers, but that the infants were 
normally proportioned. 



ZOl HD 00334-12 EB 

In ensuing studies, birth certificates of infants born in Tennessee between 1979 
to 1984 were matched with those of their mothers, who were born in Tennessee 
between 1959 to 1966. Maternal and infant birth weights were again shown to be 
correlated. In addition, women who were themselves of low birth weight were up 
to 4 times as likely to have a small-for-gestational age infant as were women who 
weighed 4000-4499 grams at birth, but low birth weight women were less than 
twice as likely to have a preterm infant. A group of Swedish women, born from 
1955 to 1965, was next studied. Women who themselves were small-for-gestational 
age at birth were at increased risk of giving birth to both small-for-gestational 
age and preterm infants. Women who were preterm at birth were not at increased 
risk of either outcome. 

Work in FY95 has yielded several findings. In the Danish study, the women had 
2046 singleton infants, 24.7% of infants born to SGA mothers were SGA; in 
comparison, 11.4% of infants born to appropriately-grown mothers were SGA 
(p<0.001). Women who were themselves preterm at birth had a 10.5% risk of giving 
birth to a preterm infant in any given pregnancy, compared to 6.6% for women born 
at term (p=0.04). SGA mothers were not at significantly increased risk of giving 
birth to a preterm infant, and preterm mothers were not at significantly 
increased risk of giving birth to a SGA infant. Adjustment for confounding 
variables did not change the results. In addition, the association between 
maternal and infant SGA varied depending on the height of the grandmother. When 
the grandmother was tall, maternal SGA was not a risk factor for infant SGA. 
However, when the grandmother was short, maternal SGA added significantly to the 
infant's risk of being SGA. Additional analyses from this study addressed the 
antecedents and effects of uncertainty of the gestational age estimate. 

Preliminary analysis of the data from Philadelphia and Providence indicates that 
the 627 interviewed women had 1131 children. Women who were themselves preterm 
at birth were not at increased risk of giving birth to a low birth weight infant 
(relative risk 1.1), but that women who were small-for-gestational age at birth 
were at significantly increased risk of giving birth to a low birth weight infant 
(relative risk 1.7, p<0.05). Data regarding infant gestational age are currently 
being edited. 

Significance to Biomedical Research and the Program of the Institute ; This 
project suggests that the pathogenesis of low birth weight begins even before the 
birth of the mother. This implies that future research on prevention and 
management of at risk pregnancies should take into account the intrauterine and 
perinatal experience of a mother. It also implies that changes in the incidence 
of preterm delivery and intrauterine growth retardation may take several 
generations to accomplish. Further research is needed to determine the relative 
contribution of genetic effects versus environmental factors (e.g. smoking) 
passed across generations on low birth weight. 

Proposed Course of Project : Data analysis is ongoing and several manuscripts are 
being drafted. 

The Tennessee data were published in the Journal of Pediatrics . The results from 
Sweden have been published in Pediatrics . A paper describing the Danish study 
methods was published in Paediatric and Perinatal Epidemiology . 



ZOl HD 00334-12 EB 

Further results of this project will be published in peer-reviewed journals. 

Publications : Previously listed. 

Presentations ; Preterm and small-for-gestational age birth across generations 
in U.S. whites and blacks. American Public Health Association, 1992. 

Uncertain gestational age - a souce of bias in epidemiologic studies. Society 
for Pediatric Epidemiologic Research, 1995. 

SGA and preterm birth across generations. Society for Pediatric Epidemiologic 
Research, 1995. 

Contract Report ; 

This project has been supported by the University of Pennsylvania, under NICHD 
contract Number NOl-HD-7-2909, and by the University of Southern California, 
under NICHD contract Number NOl-HD-7-2902. These contracts, both entitled 
"Preterm and Small for Gestational Age Birth Across Generations," were initiated 
on 3-1-87 and 5-1-87, respectively, and terminated during FY92. The main 
objectives of these contracts have been to locate, interview and abstract the 
obstetrical records of a group of women who were themselves preterm, small for 
gestational age or normal-sized at birth. The methods employed included 
interviews with human subjects, and medical record abstraction. During the 
current fiscal year, interviewing was completed, medical record abstractions have 
been completed, and data processing and analysis are ongoing. The Project 
Officer has been extensively involved in the design and execution of these 
contracts. He designed the sample selection procedures and had primary 
responsibility for designing the study forms. In addition, he closely monitored 
the subject location procedures and was responsible for developing solutions to 
aid in increasing the follow-up rate. He is also responsible for carrying out 
the data analysis. This contract is essential for locating and interviewing the 
appropriate research subjects, because such women are otherwise unavailable to 
NICHD researchers. 



DEPARTMBfl' OF HEALTH AND HUMAN SSRVICES - PUBLIC HEALTH SERVICe 

NOTICE OF INfTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00361-09 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (30 characters or less. Title must fit on one line between the borders.) 

Child Health Supplement to the 1988 National Health Interview Survey 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 



Other: A. Trumble 



Acting Branch Chief 



CSB, DESPR, NICHD 



COOPERATING UNITS (if any) 

HLB, CRMC, NICHD (P. C . Scheldt ) ; University of North Carolina at Chapel Hill 
(J.Kotch); Bar Ilan University (Y.Harel); Centers for Disease Control (D.Jones); 
Albert Einstein College of Medicine (P.Bijur) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



1.1 



PROFESSIONAL: 



1.0 



0.1 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects 
D (a1) Minors 
(a2) Intervievi/s 



D (b) Human tissues 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

NICHD sponsored and coordinated the 1988 survey to provide population-based data on 
children's health. Early work focused on effects of low birth weight and maternal 
smoking on children's later health status. The later work has analyzed childhood 
injury risk factors to support effective targeting of interventions since injury is 
the leading cause of death for children in the U.S. This is the first study to 
demonstrate that non-fatal injury causes differed considerably from fatal causes, 
with considerable variation of risk factors by age and socioeconomic 
characteristics. Analysis of injuries in preschool children by child care patterns 
showed that use of non-guardian care did not increase risks except for the youngest 
children of poor, low education mothers who use multiple part-time places of care 
of all types (center-based, care in their own home or homes of others). Increasing 
hours in center-based care seemed to lower both total and serious injury risks for 
children ages 4-5 years. Also, older children of women who are problem drinkers, 
or of two parents who are both problem drinkers, are at higher injury risk. 
Another risk factor analysis showed that sports and recreation account for 36Z of 
all medically attended injuries from all causes. Analysis of effects of access to 
medical care showed that for children without medical care coverage (health 
insurance or Medicaid), as much as 30Z of total injuries and 40Z of serious 
injuries may not have received medical attention compared to children with medical 
care coverage. The latest studies have shown that children in single adult 
households are at increased risk regardless of other factors such as poverty or 
race . 



PHS 604C (Rev. 5/92) 



ZOl HD 00361-09 EB 

Project Description 

Objectives : 1) To provide prevalence estimates of health status Indicators, 
Injuries, child care, and family structure for all U.S. children. 2) To learn 
the determinants of children's health and Injury risks. 

Methods Employed ; During 1988 NCHS conducted an interview survey of a 
representative sample of all households in the U.S. as part of the continuous 
National Health Interview Survey. About 17,200 sample children were selected. 
Questions pertained to the child's perinatal and birth period, family structure, 
child care, health status and behavior. The questionnaire was developed by a 
cooperative effort from representatives of the NICHD, U.S. Health Resources and 
Services Administration, National Center for Health Statistics and Child Trends, 
Inc. The Epidemiology Branch of NICHD had major responsibility for coordination. 

Major Findings : 1) No effect on Injury risk for preschool children was found for 
most nonguardian child care use. However, risk of serious injury decreased with 
Increasing hours in center-based care at ages 4-5 years. Serious Injury risk 
Increased for children <2 years old whose mothers had <12 years of education and 
who used part-time and multiple places of care across all types of care combined 
compared to using either full-time or no care. 

2) Twelve-month recall of medically attended Injuries is affected by severity of 
the Injury, age and sex of the child, and the type of Injury event. 

3) Non-fatal Injuries show a different epidemiological pattern from fatal 
Injuries reflecting interaction of types of exposures and developmental stages. 

4) Children of women who are problem drinkers have an elevated injury risk; 
children with two parents who are problem drinkers are at higher risk. 

5) Parents of children without either health insurance or Medicaid are less 
likely to report medically attended injuries, regardless of Injury severity. 
Studies based on Injury reports from treatment sources could be biased when 
assessing the effects of exposures associated with lack of medical coverage. 

6) Sports injuries account for 36% of all medically-attended injuries. Cause and 
nature of Injury are strongly related to age. 

7) Children living in single parent households are 40 percent more likely to be 
injured than those in two-parent households. 

Significance to Biomedical Research and the Program of the Institute : This 
survey documents current health status and its relation to accidents, injuries, 
poisonings, exposure to cigarette smoke, early health conditions, family 
structure, child care, and use of health services and behavior. Normative ranges 
are established. The data enable prioritizing of targeted interventions and 
research for children through representative U.S. population-based estimates. 

Proposed Course : Three final analyses on Injury risks associated with child 
care and socioeconomic factors are under review for submittal or publication. 



ZOl HD 00361-09 EB 

Publications : 

Overpeck MD, Kotch JB. Effects of access to care on medical attention for 
injuries. Am J Publ Hlth 1995;85:402-4. 

Overpeck MD, Trumble AC, Brenner RA. Population-based surveys as sources of U.S. 
injury data and special methodological problems. Proceedings of the 
International Collaborative Effort on Injury, Bethesda, MD, May 18-20, 1994. 
Hyattsville, MD: DHHS Pub. No. (PHS) 95-1252;12-7. 

Scheidt PC, Harel Y, Trumble AC, Jones DH, Overpeck MD, Bijur PE. Epidemiology 
of non-fatal injuries in children and youth. Am J Publ Hlth 1995;85:932-8. 

Harel Y, Overpeck MD, Jones DH, Schedit PC, Bijur PE, Trumble AC. The quality 
of proxy-respondent data m NCHS surveys. [Letter to the Editor]. Am J Publ 
Hlth 1995;85:591. 

Overpeck MD, Moss AJ. Wease; words?: passive reporting on passive smoke. 
[Letter to the Editor.] MediaCritic 1994;2:104-5. 

Overpeck MD, Moss AJ. Smoking studies. [Letter to the Editor.] Los Angeles 
Times, August 3, 1994. 

Bijur PE, Trumble AC, Harel Y, Overpeck MD, Jones DH, Scheidt PC. Sports and 
recreation injuries in U.S. children and youth. Arch Pediatr Adolesc Med, in 
press. 



10 



DEPAFrTMEhfT C3F HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SEFWICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00369-07 EB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must m on one line between the borders.) 

Adverse Perinatal Events and Subsequent Injury-related Death 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 



PI: M.A. Klebanoff 



Research Medical Officer 



EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

NIMH (M. Farmer) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.05 



PROFESSIONAL: 



0.05 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
n (a2) Interviews 



D (b) Human tissues 



B (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Several published studies have indicated that children experiencing asphyxia during 
the perinatal period are at increased risk of subsequent adolescent suicide. 
However, these studies usi.d retrospectively ascertained data and were unable to 
control for the adverse social conditions that often accompany perinatal 
difficulties . 

In the first phase of this project, the names of the approximately 55,000 children 
who were born during 1959-66 as subjects in the Collaborative Perinatal Project 
were computerized. During the second phase, the computerized names and other 
appropriate identifying information were submitted to the National Death Index, and 
all subjects who have died were identified. In the third phase, the death 
certificates of these subjects will be obtained and cause of death recorded. Since 
the Collaborative Project collected extensive data about the subjects' prenatal, 
perinatal, and childhood histories, it will be possible to study prospectively the 
relationship between adverse perinatal events and subsequent risk of death, as well 
as cause of death. 



PHS 6040 (Rev. 5/92) 



1. 



ZOl HD 00369-07 EB 

Project Description 

Objectives : The purpose of this project is to evaluate the relationship between 
adverse perinatal events and subsequent rates and causes of death in a population 
with thoroughly documented prenatal, perinatal and childhood histories. 

Methods Employed : This project will utilize individuals who were born as 
subjects in the Collaborative Perinatal Project, a multicenter study of 
pregnancy, delivery and childhood that prospectively enrolled approximately 
60,000 pregnancies from 1959-66. The subjects' names, which do not exist on 
computer files, were entered onto a computer. This task was completed in the 
winter of 1993. Following this, the names, along with other required identifying 
information, were submitted to the National Death Index, where it was possible 
to match them to all deaths in the United States from 1979 to the present. 
Results were obtained in the spring of 1994. For subjects who have died, the 
death certificate will be obtained and cause of death coded. Rates and causes 
of death will be compared to determine the effect of perinatal asphyxia on 
subsequent adolescent and young adult mortality. 

Major Findings : None yet. 

Significance to Biomedical Research and the Program of the Institute : This 
project will provide valuable additional data on the relationship between 
perinatal events and subsequent young adult death, and will enable the 
intervening role of adverse social situation to be investigated. The project 
will add significantly to the present state of knowledge about the long term 
effects of perinatal events. 

Proposed Course of Project : The preparation of the data tape has been completed. 
The tape was submitted to the National Death Index in the winter of 1993, and 
results obtained in the spring of 1994. Death certificates of possible matches 
will be obtained using FY96 funds. 

Publ ications : None yet 



X(^ 



DEPARTMEMT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SEFWICE 

NOTICE OF im"RAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00373-07 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.} 

Calcium Supplementation in Pregnancy for the Prevention of Preeclampsia 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: R.J. Levine Expert (Epidemiology) EB, DESPR, NICHD 



Others: R. DerSimonian 
J.D. Clemens 
M.A. Klebanoff 



Mathematical Statistician 

Branch Chief 

Research Medical Officer 



BMSB, DESPR, NICHD 
EB, DESPR, NICHD 
EB, DESPR, NICHD 



COOPERATING UNITS (If any) 

University of Alabama (J.C.Hauth); Case Western Reserve U. (P.M.Catalano) ; Univ. of 
New Mexico (L.B.Curet); Oregon Health Sci.Univ. (C.Morris); Univ. of Tennessee 
(B.M.Sibai); The Emmes Corp. ( J.Esterlitz) ; Biomedical Research Inc. (J. Leaf) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda , Maryland 20892 



TOTAL STAFF YEARS: 



1.4 



PROFESSIONAL: 



1.4 



CHECK APPROPRIATE BOX(ES) 

B (a) Human subjects 
E (a1) Minors 
B (a2) Interviews 



B (b) Human tissues 



□ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The results of ten clinical trials suggest that supplemental calcium may prevent 
preeclampsia. Methodologic problems, however, and differences in study design 
limit the credibility of the results and their generalizability to other patient 
populations. Moreover, none of the trials has reported the outcome of systematic 
surveillance for urolithiasis, an important possible complication of treatment. In 
response to the need for a definitive evaluation of the effects of calcium 
supplementation, NICHD is conducting a trial at five U.S. university medical 
centers. Healthy nulliparous patients are randomly assigned to receive either 2 g 
ingested supplemental calci'im daily (n=2250) or placebo (n=2250) in a double-blind 
study. Medication is administered beginning between 13 and 21 completed weeks of 
gestation and continued until the termination of pregnancy. Monitoring for the 
major study endpoints - pregnancy-associated hypertension and proteinuria, 
preeclampsia, eclampsia, and HELLP syndrome - and for urolithiasis will be 
systematic, standardized, and thorough. It will include measurement of blood 
pressure, proteinuria, and hematuria at uniformly scheduled prenatal clinic visits 
and surveillance for hypertension and proteinuria during labor, delivery, and the 
first 24 hours postpartum. CPEP should have adequate statistical power to detect a 
reduction of 43 percent in preeclampsia risk in the calcium group. 



PHS 6040 (Rev. 5/92) 



ZOl HD 00373-07 EB 

Project Description 

Objectives : The purpose of this investigation is to evaluate the efficacy of 2 
gm/day ingested supplemental calcium for reducing the incidence of important 
hypertensive disorders of pregnancy - preeclampsia, eclampsia, and HELLP syndrome 
- in healthy nulliparous women. 

Methods Employed : The study is a multicenter , randomized, double-blind, placebo- 
controlled clinical trial. Besides nulliparity, study subjects lack known risk 
factors for preeclampsia. Blood pressure is monitored at prenatal clinic visits 
by trained nurses using mercury sphygmomanometers and cuffs of appropriate size. 
Proteinuria is measured by total protein excretion in 24-hour urine collections 
and in random urine specimens, by dipsticks and by protein/creatinine ratios. 

Major Findings ; None yet. 

Significance to Biomedical Research and the Program of the Institute : Should 
calcium supplementation be proven beneficial, it may become a part of routine 
prenatal care with the purpose of reducing the risk of hypertensive disorders of 
pregnancy and decreasing associated maternal and perinatal morbidity and 
mortal ity. 

Proposed Course of Project : The field trial was launched in June 1992, following 
a one-month pilot phase. During FY 1996 the follow-up of study subjects will be 
completed, and the database cleaned and frozen for analysis. Site visits will 
be conducted, and a joint meeting of the Steering Committee and the Data Safety 
and Monitoring Board will be convened at which the results of the study will be 
presented. 

Publications : Previously listed. 

Contract Report ; The project has been supported by The Emmes Corporation, the 
University of Alabama, Case Western Reserve University, the University of New 
Mexico, Oregon Health Sciences University, the University of Tennessee, and 
Biomedical Research Incorporated under the following respective contract numbers; 
NOl-HD-1-3121, NOl-HD-1-3122, NOl-HD-1-3125, NOl-HD-1-3124, NOl-HD-1-3123, NOl- 
HD-1-3126, and NOl-HD-2-3154. During FY95 $2,373,702 has been allotted to these 
contracts. The major objective of the contracts is to provide support to five 
clinical centers, which will enroll and follow patients for the trial; to a data 
coordinating center, which will be responsible for all data management and 
analysis; and to a central repository for storage of laboratory specimens 
obtained from study subjects. During the current fiscal year patient enrollment 
has been completed. The collection of data obtained during patient follow-up 
continues. The above contracts are essential since NICHD does not have 
facilities for enrolling and monitoring pregnant women, sufficient statistical 
staff to maintain a data coordinating center, or the facilities and personnel 
necessary for operating an ultra-low temperature storage facility. 



1 



DEPAHTMENT OF HEALTH AND HUMAN SEFWICES - PUBLIC HEALTH SERVK3E 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00379-06 EB 



PERIOD COVERED 



October 1, 199A through September 30, 1995 



TITLE OF PROJECT (60 characters or less. Title must fit on one line between the borders.) 

Data Analysis from the Vaginal Infections and Prematurity Study 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: M.A. Klebanoff Research Medical Officer EB , DESPR, NICHD 



COOPERATING UNITS (if any) 

PAMA, CRMC, NICHD (R.Nugent); NIAID (M.F.Cotch); Research Triangle Institute 
(A.V.Rao); Columbia University (J.Regan) 



LAB/BRANCH 

Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



PROFESSIONAL: 



.3 



OTHER: 



CHECK APPROPRIATE eOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The Vaginal Infections and Prematurity (VIP) Study, jointly sponsored by NICHD and 
NIAID, is a prospective study designed to investigate the relationship between 
genital tract colonization with various microorganisms and the subsequent 
development of preterm birth. It also incorporates a clinical trial of 
erythromycin to prevent preterm birth among women colonized with Ureaplasma 
urealyticum. Chlamydia trachomatis, and group B streptococcus. In addition, the 
VIP collected a wealth of additional data on a variety of factors possibly linked 
to pregnancy outcome. This project will analyze these factors. 

Analyses undertaken to date include the predictive value of vaginal Gram stain in 
the identification of group B streptococcus, the descriptive epidemiology of group 
B streptococcal carriage, the relationship between reported physical activity and 
preterm birth, the effect of treatment with erythromycin on pregnancy outcome among 
women colonized with group B streptococci, and the association between sexual 
intercourse during pregnancy and preterm birth among colonized with different 
genital microorganisms. Questions under analysis include the effect of group B 
streptococcal colonization on pregnancy outcome, the association between bacterial 
vaginosis and pregnancy outcome, and the effect of treatment with erythromycin on 
pregnancy outcome among women colonized with Chlamydia trachomatis. 



PHS 6040 (Rev. 5/92) 



Ic; 



ZOl HD 00379-06 EB 

Pro.ject Description 

Objectives : The objective of this project is to investigate factors associated 
with adverse pregnancy outcome in the Vaginal Infections and Prematurity (VIP) 
data set. 

Methods Employed ; This study, sponsored by NICHD and NIAID, is a prospective 
study designed to investigate the relationship between genital tract colonization 
with various microorganisms and the subsequent development of preterm birth. It 
also incorporates a clinical trial of erythromycin to prevent preterm birth among 
women colonized with Ureaplasma urealyticum, Chlamydia trachomatis, and group B 
streptococcus (GBS). In addition, the VIP collected a wealth of additional data 
on a variety of factors possibly linked to pregnancy outcome. 

Major Findings : Several analyses have been conducted to date. The utility of 
a cervicovaginal Gram stain as a screening tool for carriage of group B 
streptococcus was evaluated. The sensitivity, specificity, positive and negative 
predictive values were 28, 69, 17, and 81% at mid gestation, and 34, 72, 18, and 
86% at delivery. The positive and negative predictive values differed little 
from the baseline prevalence of the organism. It was concluded that the Gram 
stain was not a useful screening tool for the detection of group B streptococcal 
carriage. 

An additional analysis detailed the descriptive epidemiology of group B 
streptococcal colonization during pregnancy. Various factors associated with 
group B streptococcal carriage were described. For example, group B 
streptococcus was more common among older women and women of lower parity; it was 
less common among women currently living with their partner, and less common 
among current smokers. Increasing years of sexual experience resulted in a 
decreased risk of colonization. Group B streptococcus was present more 
frequently among women colonized with Candida species, but group B streptococcus 
was not more common among women colonized with Chlamydia, Ureaplasma or 
Trichomonas, compared to women not colonized with these organisms. 

The analysis of the relationship between group B streptococcal carriage and 
pregnancy outcome is ongoing. Preliminary analyses do not demonstrate a 
significant association between carriage of the organism and either preterm 
delivery or low birth weight. Similarly, these adverse outcomes were not more 
common among women heavily colonized with group B streptococcus than among those 
lightly colonized. However, further analysis demonstrated that nearly 25% of 
women received a variety of antibiotics for clinical indications during the third 
trimester. Among women who did not receive clinically indicated antibiotics, 
increasing density of streptococcal colonization was associated with a 
significantly increasing risk of delivery before 32 weeks, as well as with a 
significantly increasing risk of giving birth to a low birth weight (<2500 gram) 
or very low birth weight (<1500 gram) infant. Results from the clinical trial 
of erythromycin indicate that treatment of streptococcal carriers with 
erythromycin does not result in a reduced occurrence of early delivery or 
delivery of a low birth weight infant. 



iu 



ZOl HD 00379-06 EB 

A fourth analysis investigated the relationship between reported physical 
activity and preterm birth. Prolonged periods of standing were modestly 
associated with preterm birth (odds ratio for >8 hours/day of standing = 1.31). 
Heavy work/exercise was not associated with preterm birth (odds ratio for >4 
hours/day of heavy work = 1.04). The proportion of infants born preterm did not 
differ among women employed in predominantly standing, active or sedentary 
occupations. Physical activity was not associated with gestational age-adjusted 
birth weight. In an updated analysis, prolonged standing was no longer 
associated with preterm birth. 

An additional analysis evaluated the association of reported frequency of sexual 
intercourse at 23-26 weeks and preterm birth. Among all women, sexual 
intercourse was associated with a 20% reduction of preterm birth. However, the 
effect of sexual intercourse was dependent on the presence of certain micro- 
organisms. Among women who were not colonized with Trichomonas vaginalis, sexual 
intercourse was associated with a reduced risk of preterm birth, but among women 
who were colonized with this organism, intercourse was not associated with 
preterm birth. Similar results were obtained for colonization with Mycoplasma 
hominis. Conversely, T. vaginalis and M. hominis were risk factors for preterm 
birth only among women reporting frequent sexual intercourse. 

In another analysis, the association between the presence of bacterial vaginosis 
and pregnancy outcome was studied. Bacterial vaginosis was present in 16% of the 
study women, and was associated with the delivery of a preterm-low birth weight 
infant (odds ratio 1.4, 95% confidence interval 1.1-1.8). Metronidizole use 
between study enrollment and delivery was associated with a decrease in the 
occurrence of preterm-low birth weight. 

Significance to Biomedical Research and the Program of the Institute : 
Preliminary results from the clinical trial indicate that treatment of all women 
colonized with group B streptococcus is unlikely to reduce their risk of preterm 
or low birth weight delivery. It is possible, however, that treatment of the 
more densely colonized women might have some benefit. These analyses indicate 
that Gram stain is not an effective tool to screen for group B streptococcal 
carriage, implying that other screening methods for this important perinatal 
pathogen need to be devised. The results of the analysis of the relationship 
between activity and preterm birth have both clinical and research implications. 
Practical implications are that for most women with uncomplicated pregnancies, 
there is no need to restrict activity in the hopes of preventing preterm birth. 
Important research implications are that on a population basis, changes in 
maternal activity during pregnancy are not likely to have large effects on the 
rate of preterm birth, and that unmeasured differences in socioeconomic status 
between women reporting different amounts and types of physical activity were 
likely to have accounted for previous positive results on this subject. 

The results of the analysis of sexual intercourse shed important new light on the 
role of infection in the pathogenesis of preterm birth. First, they imply that 
for most pregnant women, intercourse does not adversely affect the pregnancy. 
Second, the results suggest that when certain organisms are present in the 
vagina, their introduction into the cervix (as might happen during intercourse) 
can lead to preterm labor. 



J. f 



ZOl HD 00379-06 EB 

The analyses of bacterial vaginosis have identified an important, treatable risk 
factor for preterm birth. 

Proposed Course of Pro.iect : The analysis of group B streptococcal colonization 
on pregnancy outcome is continuing under review. 

Publications ; 

Klebanoff MA, Regan JA, Rao AV, Nugent RP, Blackwelder WC, Eschenbach DA, 
Pastorek JG, Williams S, Gibbs RS, Carey JC for the Vaginal Infections and 
Prematurity Study Group. Outcome of the vaginal infections and prematurity 
study: results of a clinical trial of erythromycin among pregnant women colonized 
with group B streptococci. Am J Obstet Gynecol 1995;172:1540-5. 

Hillier SL, Nugent RP, Eschenbach DA, Krohn MA, Gibbs RS, Martin DH, Edelman R, 
Cotch MF, Pastorek J, Rao AV, McNeills D, Regan JA, Carey JC, Klebanoff MA. The 
association of bacterial vaginosis, bacteroides, and Mycoplasma hominis with 
preterm, low birth weight delivery. N Engl J Med, in press. 



10 



DEPARTMENT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SEFMCE 

NOTICE OF l^^"RAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00382-05 EB 



PERIOD COVERED 



October 1, 199^ through February 28, 1995 



TITLE OF PROJECT (80 characters or tess. Title must fit on one line between the borders.) 



Cocaine and Marijuana Use During Pregnancy and Pregnancy Outcome 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) [Name, title, laboratory, and Institute affiliation) 



PI: M.A. Klebanoff 



Research Medical Officer 



EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Office of the Director, DESPR (H.W.Berendes ) ; PAMA, CRMC (R.Nugent); Research 
Triangle Institute (M.Cotrh); Packard Foundation (P.Shiono); University of Utah 
(D. Rollins) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



,20 



PROFESSIONAL: 

.20 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n {a1) Minors 
□ (a2) Interviews 



B (b) Human tissues 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 



This project is terminated. 



Vj 



PHS 6040 (Rev. 5/92) 



DEPARIMENT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SEFMCE 

NOTICE OF irJTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00383-05 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. We must fit on one line between the borders.) 

Analyses of Data from the Collaborative Perinatal Project 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: M.A. Klebanoff Research Medical Officer EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

University of California at San Francisco (T.Newman); NEI (E.Chew); 
University of Maryland (D.Nagey); Packard Foundation (P.Shiono) 



LAS/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



,15 



PROFESf;,ONAL: 



.15 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
n (a2) Interview/s 



B (b) Human tissues 



D (c) Neitlier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The Collaborative Perinatal Project (CPP) represents a rich data set for the 
investigation of a variety of topics related to maternal and child health. 

Currently active areas of investigation include the descriptive epidemiology of 
congenital cataract, and the association between elevated maternal serum levels of 
alpha fetoprotein at 14-20 weeks and subsequent stillbirth. 



PHS 6040 (Rev 5/92) 






ZOl HD 00383-05 EB 
Pro.ject Description 
Ob.iectives ; 

1. To determine the association between neonatal hyperbilirubinemia and 
neurodevelopmental abnormalities at age 7-8. 

2. To state the descriptive epidemiology of strabismus and congenital cataract. 

3. To determine the association of elevated maternal serum alpha fetoprotein at 
14-20 weeks and subsequent stillbirth. 

4. To determine whether perinatal exposure to vitamin K is associated with an 
increased risk of cancer during childhood. 

Methods Employed ; The Collaborative Perinatal Project (CPP) enrolled pregnant 
women entering prenatal care at 12 university hospitals across the United States 
from 1959 to 1966. The women were followed throughout pregnancy, labor and 
delivery, and their children received multiple physical, neurological and 
psychological exams during their first 7 to 8 years of life. Maternal serum was 
collected at multiple times during pregnancy. Extensive data were collected on 
a wide variety of obstetric and pediatric conditions. 

One investigation using this dataset completed during FY94, in collaboration with 
Dr. Thomas Newman of the University of California at San Francisco, evaluated the 
association between neonatal hyperbilirubinemia and neurodevelopmental outcome 
at age 7-8. The CPP required that serum bilirubin be measured on every neonate 
according to a fixed protocol. Extensive psychologic testing was performed at 
age 7, as was a detailed neurologic examination. In addition, a smaller group 
of children underwent tests of speech and hearing function at age 8. This 
analysis compared the Wechsler IQ at age 7 of children with different values of 
peak neonatal serum bilirubin. In addition, the presence of an abnormal 
neurologic examination at age 7 was studied among children with different peak 
bilirubin values. Finally, hyperbilirubinemic and non-hyperbilirubinemic 
children were compared for the presence of sensorineural hearing loss at age 8. 
The benefits of treating hyperbilirubinemia in otherwise healthy term infants are 
controversial. Since the clinical management of healthy term infants has changed 
little over the past decades, data of this age are still relevant. In fact, the 
absence of phototherapy as a treatment for hyperbilirubinemia makes these data 
even more valuable, as it would be very difficult to assemble a cohort of 
neonates with untreated hyperbilirubinemia today. 

A second investigation, done in collaboration with personnel at the Epidemiology 
Branch of the National Eye Institute, is addressing the descriptive epidemiology 
of strabismus and congenital cataract. This condition is relatively common, 
affecting 2-3% of all children. Review of the literature reveals few studies 
describing risk factors for strabismus. The CPP conducted detailed neurologic 
examinations at ages 1 and 7, making it well-suited for the study of this 
condition. 



ZOl HD 00383-05 EB 

A third investigation, done in collaboration with Dr. David Nagey of the 
University of Maryland, evaluated the predictive value of elevated maternal serum 
alpha fetoprotein for stillbirth in otherwise normal infants. In this study 
alpha fetoprotein levels in serum drawn at 14-20 weeks gestation from mothers 
delivering non-malformed stillborn infants were compared to levels in women 
delivering liveborn infants. Each stillborn case was matched to 4 liveborn 
controls for study site, race, gestation at which blood was obtained {±3 days), 
and years elapsed since the serum was drawn {+2 years). The fraction of cases 
and controls with alpha fetoprotein levels of greater than 3 multiples of the 
median was compared. 

A fourth investigation studied the risk of childhood cancer following perinatal 
vitamin K exposure. Two previous reports have indicated that children who 
received this drug during the neonatal period are at approximately twofold 
increased risk of developing cancer. Since nearly every child born in the United 
States during the past several decades has received intramuscular vitamin K, up 
to half of all childhood cancer might be attributable to the drug. The CPP is 
an ideal cohort in which to study this issue, as it spanned the era when vitamin 
K exposure changed from being rare to being common. 

Ma.jor Findings : Peak serum bilirubin was not associated with 7-year IQ in either 
black or white infants. Among white infants the IQ of children whose bilirubin 
value was >20 mg/dl was 105.0, compared to 103.4 for children whose bilirubin 
value was <20 mg/dl. The corresponding values for black children were 91.0 and 
93.3. Definitely abnormal neurologic examinations at age 7 were not more common 
among children whose peak neonatal bilirubin value was >20 mg/dl (4.5%) compared 
to those who were <20mg/dl (3.8%). However, the frequency of abnormal or 
suspicious neurologic examinations increased in a stepwise manner from 14.9% in 
children with bilirubin values <10 mg/dl to 22.4% in those with peak bilirubin 
values of >20 mg/dl (p<0.001). This increase was accounted for by minor, non- 
specific motor abnormalities. 

In the alpha-fetoprotein analysis, the odds ratio for fetal death in pregnancies 
with serum alpha-fetoprotein values >3.0 multiples of the median was 1.98 
(p=0.018). However, the sensitivity of elevated alpha fetoprotein for prediction 
of fetal death was only 8.5%; the specificity was 95.7%. In a modern-day 
population with a probability of late fetal death of 1%, the predictive value of 
a positive test would be only 2%, which is not sufficiently high to be of 
clinical utility. 

There were 48 children in the CPP who developed cancer after the first day of 
life, including 16 who developed leukemia. This resulted in a cumulative 
incidence of cancer and leukemia of 1.1 and 0.4 per 1000, respectively, by age 
7-1/2. Neonatal exposure to vitamin K was found not to be associated with the 
development of cancer during childhood (odds ratio 0.84, 95% confidence interval 
0.41 to 1.71), or of leukemia (odds ratio 0.47, 95% confidence interval 0.14 to 
1.55). 



ZOl HD 00383-05 EB 

The results of the strabismus analyses indicate that 3% of the cohort developed 
esotropia and 1.2% developed exotropia. Esotropia was approximately twice as 
common in white as in black children, but exotropia did not differ by race. Both 
types of strabismus were more common in low birth weight infants. Maternal 
cigarette smoking during pregnancy was associated with an increased risk of both 
types of strabismus; this finding was independent of the birthweight-reducing 
effect of smoking. 

Significance to Biomedical Research and the Program of the Institute : Each of 
these analyses involves a problem of relevance to child health. Treatment of 
jaundice results in large health care expenditure for the United States, due to 
the frequent extension of hospital stays for otherwise normal newborns. 
Furthermore, the need for this treatment is controversial. If it were to be 
shown that physiologic hyperbilirubinemia is not harmful to normal term infants, 
management of these infar-Ls might require reevaluation. Study of risk factors 
for strabismus will further our understanding of this common condition. The 
study of serum alpha fetoprotein will demonstrate the stability of this compound 
in long-term storage. It will also determine whether or not this test is a 
clinically useful marker for predicting stillbirth. The data on vitamin K 
provide important reassurance regarding the safety of this commonly used drug. 

Preliminary results of the cataract analysis revealed few risk factors for the 
condition - other than low birth weight. Ongoing analysis will evaluate the 
association between cataract and other congenital anomalies. 

Proposed Course of Project ; Data analyses of the cataract and alpha-fetoprotein 
results is continuing, and results will be submitted to peer-reviewed journals. 

Publications : 

Chew E, Remaley N, Tamboli A, Zhao J, Podgor M, Klebanoff M. Risk factors for 
esotropia and exotropia. Arch Ophth 1994;112:1349-55. 



.»-«» t_' 



DBPABTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SEFWICE 

NOTICE OF irfTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00384-04 EB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Field Trial of Oral Cholera Vaccines in Bangladesh 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: J.D. Clemens Branch Chief EB, DESPR, NICHD 

Other: M.R. Rao Visiting Scientist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Johns Hopkins School of Public Health (D.Sack); International Center for 
Diarrheal Disease Researc:h, Bangladesh 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.6 



PROFESSIONAL 

0.6 



CHECK APPROPRIATE BOX{ES) 

H (a) Human subjects 
H (a1) Minors 
B (a2) Interviews 



D (b) Human tissues 



n (c) Neitlier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project entails continuing analyses of data derived from a large-scale, 
placebo-controlled field i-rial of B subunit-killed whole cell (BS-WC) and killed 
whole cell-only (WC) oral vaccines against cholera, conducted between 1985-90 in 
the Matlab field studies area of the International Center for Diarrheal Disease 
Research, Bangladesh. The trial enrolled ca. 89,000 subjects for study. Analyses 
of the first three years of follow-up revealed that each vaccine conferred ca. 502 
protection against cholera episodes detected among patients seeking care at medical 
facilities. During the past year, a paper on the fourth and fifth years of 
surveillance, during which no further protection was evident, was prepared. In 
addition, an analysis of serum immune responses to V^ cholerae 01 among vaccine 
failures revealed that such vaccinees manifested poor responses, suggesting that 
impaired immunity may play a role in instances of vaccine failure. 

During the past year, a large-scale case-control study was conducted to determine 
whether infection by Helicobacter pylori . a known cause of persistent 
hypochlorhydria , is a risk factor for cholera in Bangladesh. Preliminary analyses 
indicate that the risk of clinically severe cholera is elevated in H_^ pylori - 
infected individuals, but only when such individuals lack antecedent vibriocidal 
immunity to cholera. Infection by H. pylori was not a determinant of vaccine 
failure in the field trial. Analyses of H. pylori in children indicate that both 
household crowding and Hindu religion were associated with an elevation of risk, 
but that poorer nutritional status was not a risk factor for or a consequence of H. 
pylori infection. 



PHS 6040 (Rev. 5/92) 



ZOl HD 00384-04 EB 

Project Description : 

Personnel : Dr. Clemens is the Principal Investigator of this project, with 
responsibility for the epidemiological design and execution of the study. Drs. 
Ahmed and Sack are epidemiologists who have collaborated on epidemiological 
aspects of the project. Mr. Rao has been responsible for computerized selection 
of cases and controls, and statistical analyses of the data. 

Objectives ; Specific goals of analyses conducted during the past year included: 
1) evaluation of the efficacy of the vaccines during the fourth and fifth years 
of follow-up; 2) assessment of the relationship between serum immune responses 
to infection and the likelihood of vaccine failure; and 3) evaluation of the 
relationship between infection by Helicobacter pylori and the risk of cholera. 

Methods Employed : This project entails continuing analyses of data derived from 
a large-scale, placebo-controlled field trial of B subunit-killed whole cell (BS- 
WC) and killed whole cell-only (WC) oral vaccines against cholera, conducted 
between 1985-90 in the Matlab field studies area of the International Center for 
Diarrheal Disease Research, Bangladesh. The trial enrolled ca. 89,000 subjects 
for study. After acquisition of informed consent and enrollment into the study, 
participants were randomized to receive 3 doses of BS-WC, WC, or an E . co 1 i K12 
strain placebo. Both active (community based) and passive (treatment center- 
based) surveillance techniques were employed to detect cholera infections, which 
were microbiological ly confirmed via fecal microbiology. Subjects were also 
placed under demographic surveillance, using regular visits to homes on a weekly 
or biweekly basis. Sera were drawn from cholera cases at baseline and three 
weeks later, as well as from community controls. 

To assess the relationship between H. pylori infection and the risk of cholera, 
cases with cholera were coritrasted with controls without cholera for the presence 
of serum IgG anti-H^ pylori antibodies, measured by ELISA. Both cases and 
controls were assigned to placebo in the oral cholera vaccine field trial. 

Major Findings : Analyses during the past year demonstrated that serum 
vibriocidal responses to cholera infections in vaccinees ("vaccine failures") 
were lower than those in placebo recipients during the first year of follow-up. 
These data suggest that immunological hyporesponsiveness, even to natural cholera 
infections, may have accounted for a portion of vaccine failures and imply that 
such hyporesponsiveness may set an upper limit on vaccine efficacy attainable by 
any cholera vaccine. Additional findings included: 1) The vaccines conferred 
negligible protection during the fourth and fifth year of follow-up; 2) infection 
by H. pylori was associated with the risk of clinically severe cholera, but only 
in subjects lacking natural serum vibriocidal immunity; 3) H^ pylori infection 
did not modify vaccine efficacy; and 4) crowding and Hindu religion were 
associated with a higher risk of \L_ pylori infection. 

Significance to Biomedical Research and the Program of the Institute : With the 
spread of the seventh pandemic of cholera to South America in 1991, as well as 
recognition of the increasing mortality attributable to cholera in Africa and 
Asia, the importance of developing a vaccine effective against cholera has 
recently been underscored. The findings of this trial have provided the first 
demonstration that oral, as opposed to par-enteral, vaccination is capable of 
providing long-term protection against cholera, validating human and animal data 
indicating that intestinal immunity is most relevant for protection against 
cholera and defining the direction of developmental efforts to produce new 
vaccines. 



ZOl HD 00384-04 EB 

Proposed Course of Pro.ject : The coming year will be devoted to preparing 
manuscripts on H. pylori epidemiological studies. 

Publications: 



Clemens JD, Albert MJ, Rao M, Qadri F, Huda S, Kay B, van Loon FPL, Sack D, 
Pradhan BA, Sack RB. Impact of infection by Helicobacter pylori on the risk and 
severity of endemic cholera. J Infect Dis 1995;171:1653-6. 



(^ \^' 



DEPAFrrMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SEFMCE 

NOTICE OF INTTRAMURAL RESEAflCH PROJECT 



PROJECT NUMBER 



ZOl HD 00385-04 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. We must fit on one line between the borders.) 

Epidemiology of Rotavirus Infections in Bangladesh 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: J.D. Clemens Branch Chief EB, DESPR, NICHD 

Other: M.R. Rao Visiting Scientist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

James Gamble Institute for Medical Research, Cincinnati, OH (R.Ward) 



LUe/BRANCH 

Epidemiology Branch 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.3 



PROFESSIONAL 

0.3 



CHECK APPROPRIATE BOX(ES) 

B (a) Human subjects 
(al) Minors 
n (a2) Interviews 



D (b) Human tissues 



D (c) Neittier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project tests the hypothesis that humoral immunity to rotavirus infection, 
assessed in serum, is associated with the risk of rotavirus diarrhea in children 
aged under 2 years who reside in rural Bangladesh. Cases of rotavirus diarrhea 
were assembled from population-based, comprehensive surveillance of treated 
episodes of pediatric diarrhea in Matlab, Bangladesh during 1985-86. All rotavirus 
diarrhea episodes were serotyped with monoclonal antibodies after cultivating 
rotavirus isolates. Controls were similarly aged childred randomly selected during 
four surveys of the Matlab community undertaken during 1985-86. Serological 
correlates of natural immunity to rotavirus diarrhea were assessed by contrasting 
acute-phase sera collected from the cases and sera from controls. Case-control 
comparisons revealed that: 1) serum IgG antirotavirus antibodies were correlated 
inversely with the occurrence of rotavirus diarrhea, suggesting a protective 
relationship; but 2) proteccive associations were not serotype-specif ic . As a 
second goal, we sought to determine A whether breast feeding is associated with a 
reduced risk of rotavirus diarrhea in children under the age of 2 years. 
Comparisons of cases and community controls for antecedent histories of breast 
feeding revealed that: 1) breast feeding was associated overall with a reduced risk 
of rotavirus diarrhea, due to the protection conferred by exclusive but not partial 
breast feeding; but 2) after infancy, the direction of the relationship became 
reversed, with a higher risk of rotavirus diarrhea among breast-fed than non- 
breast-fed children. 



PHS 6040 (Rev. 5/92) 



ZOl HD 00385-04 EB 
Project Description : 

Personnel : Dr. Clemens is the Principal Investigator of this project, with 
responsibility for the epidemiological design and execution of the study. Dr. 
Ahmed is an epidemiologist who has collaborated on epidemiological aspects of the 
project. Dr. Ward has conducted laboratory analyses of rotavirus isolates and 
sera. Mr. Rao has been responsible for computerized selection of cases and 
controls, and statistical analyses of the data. 

Objectives ; 

1) To evaluate whether titers of serum IgG antirotavirus antibodies are 
associated with the risk of rotavirus diarrhea in children aged under 2 years who 
reside in rural Bangladesh, and to test whether such associations are serotype- 
specif ic. 

2) To evaluate whether breast feeding is associated with a reduced risk of 
rotavirus diarrhea in children under the age of 2 years. 

Methods Employed : These analyses were designed as case-control studies. Cases of 
rotavirus diarrhea were assembled from population-based, comprehensive 
surveillance of treated episodes of pediatric diarrhea in Matlab, Bangladesh 
during 1985-86. All rotavirus diarrhea episodes were serotyped with monoclonal 
antibodies after cultivating rotavirus isolates. Controls were similarly aged 
children randomly selected during four surveys of the Matlab community undertaken 
during 1985-86. Serological correlates of natural immunity to rotavirus diarrhea 
were assessed by contrasting acute-phase sera collected from the cases and sera 
from controls. IgG antirotavirus antibodies were tested with ELISA, and serotype- 
specific antibodies were measured with plaque-neutralization assays. The 
assessment of the relationship between breast feeding and the occurrence of 
rotavirus diarrhea was based on breast feeding histories obtained by interviews 
of parents or guardians. 

Major Findings: The serological comparisons revealed the following findings: 1) 
serum IgG antirotavirus antibodies were correlated inversely with the occurrence 
of rotavirus diarrhea, suggesting a protective relationship; and 2) protective 
associations were not serotype-specif ic, but were both homologous and 
heterologous in nature. Comparisons of cases and controls for antecedent 
histories of breast feeding revealed that: 1) breast feeding was associated, 
overall, with a reduced risk of rotavirus diarrhea, due to the protection 
conferred by exclusive bjt not partial breast feeding; but 2) after infancy, the 
direction of the relationship became reversed, with a higher risk of rotavirus 
diarrhea among breast-fed than non-breast-fed children. 

Significance to Biomedical Research and the Program of the Institute : Rotavirus 
infections are the major cause of dehydrating diarrheal illnesses in infants and 
young children worldwide. The development of vaccines against these infections 
require an understanding of the basis of natural immune protection against 
rotavirus diarrhea. Serological analyses from this study reaffirm the importance 
of natural immunity in determining the epidemiological pattern of rotavirus 



9Q 



ZOl HD 00385-04 EB 

diarrhea, but question whether serotype-specif ic immunity is of central 
importance either to natural protection against rotavirus diarrhea or to vaccine- 
induced protection. Among non-vaccine strategies for the control of rotavirus 
diarrhea, breast feeding has been proposed as a potential intervention. Our data 
indicate, however, that although a biological effect of exclusive breast feeding 
was evident among infants, the reversal of a protective relationship between 
breast feeding and the risk of rotavirus diarrhea during the second year of life 
yielded no net protection among the total group of children in the study. 

Proposed Course of Project : This project has terminated. 

Publ ications: Previously listed. 






DEPARTMENT CX^ HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00386-04 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Evaluation of a Water-Sanitation Intervention in Egypt 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: J.D. Clemens Branch Chief EB, DESPR, NICHD 

Other: M.R. Rao Visiting Scientist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

UNICEF, Egypt 



LAB/BRANCH 

Epidemiology Branch 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.3 



PROFESSIONAL 

0.3 



CHECK APPROPRIATE BOX(ES) 

E (a) Human subjects 
H (a1) Minors 
(a2) Interviews 



n (b) Human tissues 



n (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The purpose of this field project is to evaluate the impact of a combined water- 
sanitation intervention on the occurrence of pediatric diarrhea and other targeted 
outcomes in a study population residing in the Assiut governorate of Upper Egypt. 

20 villages, which together included a population of approximately 10,000 persons, 
were randomly assigned to receive the intervention (N=10) or no intervention 
(N=10). The intervention consisted of provision of India Mark II tubewells and 
household latrines, together with an educational package stressing personal hygiene 
and water behaviors. To assess the impact of the intervention, all families in 
these villages were followed with logitudinal surveillance during a one-year 
follow-up period. 

Although the baseline incidence of diarrheal episodes (per 1000 person-days of 
follow-up) in children under three years of age was similar in intervention (29) 
and control (29) communities, after zero-time rates of diarrhea decreased 172 
(p<.0001) in the intervention relative to the control communities. Protective 
associations were slightly higher for bloody diarrhea (251, p<.05) and persistent 
diarrhea (26Z, p<.0001), suggesting that the intervention might have reduced not 
only the risk of diarrhea but also the severity of diarrhea. In contrast, no 
impact on the occurrence of respiratory infections was evident, indicating that the 
decline of diarrhea in the intervention communities was not likely to be due to 
ascertainment bias. It was of particular interest that intervention communities 
exhibited a marked increase of per capita water consumption, but that the new pumps 
continued to produce water that was contaminated by fecal coliforms. There data 
suggest that the salutory effect of the intervention may have been mediated, in 
part, by increased water consumption, though the water itself was fecally 
contaminated. Analyses are now continuing. 



PHS 6040 (Rev. 5/92) 



:U 



ZOl HD 00385-04 EB 

Pro.iect Description : 

Personnel : Dr. John Clemens of the Epidemiology Branch, DESPR, directs this 
study. Subjects were collected under contract by UNICEF to SPAAC, a private 
research firm in Cairo. Mr. Mai la Rao of the DESPR is conducting the analysis. 

Objectives : 

1. To assess whether an intervention intended to provide tubewells and family 
latrines, as well as water-hygiene education, can improve salutary water- 
sanitation behaviors, as well as other measures of sanitation, such as 
environmental cleanliness, microbiological purity of stored water, and overall 
water usage. 

2. To assess whether the intervention also results in a diminution in the rate 
of pediatric diarrhea in targeted villages. 

Methods Employed : After assembly of consenting families and acquisition of 
informed consent, participating villages were randomly allocated, using 
stratified randomization, to intervention an control groups. Following 
implementation of the intervention, all participating families were visited on 
a weekly basis, and, at varying intervals, measurements of target outcomes were 
made with use of direct questionnaires, observations of relevant behaviors in the 
home environments, and observations of water use at the pumps, microbiological 
sampling of water in the homes and at the pumps. 

Major Findings : The major finding obtained during the fiscal year was that 
children in the intervention villages experienced a 17% reduction in the rate of 
diarrhea, an effect that applied to both watery and non-watery diarrhea, and to 
acute and persistent diarrhea. Importantly, no protection was observed against 
acute respiratory infections, as would be predicted on epidemiological grounds. 
Moreover, the prevention of diarrhea associated with the intervention occurred 
despite increased per capita consumption of fecal ly contaminated water in the 
intervention communities. 

Significance to Biomedical Research and the Program of the Institu te: Pediatric 
diarrhea constitutes a major source of morbidity and mortality worldwide. 
Efforts to control pediatric diarrhea have, in the past, focused largely on 
improved case management, largely through promotion of oral rehydration therapy. 
However, these efforts have done relatively little to control the problems of 
dysentery and persistent diarrhea, for which preventive measures may be 
necessary. This evaluation of a pragmatic intervention which is already being 
incorporated into public health strategies by UNICEF, demonstrates that a 
multipronged water-sanitation program may indeed be capable of complementing the 
salutary effects of the case-management strategy. 

Proposed Course : During the coming fiscal year, final analyses of the impact 
upon both proximate (e.g., behavioral, environmental) and ultimate outcomes 
(pediatric diarrhea and nutritional status) will be conducted. The project will 
also provide a rich set n-f data to address several issues in diarrheal disease 



'J 
Q J- 



ZOl HD 00386-04 EB 

epidemiology, such as the impact of breast feeding on the risk of diarrhea, and 
the interaction of this association with environmental cleanliness; and the 
relationship between water use and water cleanliness with risk of pediatric 
diarrhea. 

Publications: None 



Of, 



DEPAPnrMEhfT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SEFMCE 

NOTICE OF im"RAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00389-04 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 



Studies of the Epidemiology of Pediatric Shigellosis in Bangladesh 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: J.D. Clemens Chief EB , DESPR, NICHD 

Other: M.R. Rao Visiting Scientist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

International Center for Diarrheal Disease Research, Bangladesh; Johns Hopkins 
University (F.Ahmed) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

.20 



PROFESSIONAL 

.20 



CHECK APPROPRIATE BOX(ES) 

B (a) Human subjects 
B (a1) Minors 
(a2) Interviews 



n (b) Human tissues 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The purpose of this field project is to evaluate the epidemiological patterns and 
determinants of pediatric shigellosis occurring among residents in a research field 
site in rural Bangladesh who have had a documented recent exposure to a neighbor- 
hood resident having Shigella dysentery. 1934 children under 5 years of age, from 
240 neighborhoods having a resident who presented for treatment of documented 
Shigella dysentery within 1 week of the initiation of active neighborhood surveil- 
lance, were assembled. Each of these children was followed with intensive active 
surveillance for one month, with a systematic schedule to ascertain symptomatic 
histories related to diarrhea, and to collect fecal specimens for microbiological 
characterization. Analyses have demonstrated that the occurrence of Shigella 
diarrhea was related to age (being highest in the third year of life), nutritional 
status (being higher in stunted children), season (being lowest during the monsoon 
season in the summer months), and breast-feeding status (being lower among breast- 
fed children, even up to 36 months of age). The presence of a latrine was not 
related to the risk, due to the fact that the presence of an unsanitary hanging 
latrine elevated the risk, while the presence of a sanitary latrine slightly 
depressed the risk. The lower risk of shigellosis in breast-fed than in non-breast 
fed children was almost entirely attributable to the increase in risk of shigrlosis 
which occurred during the initial 3 months following cessation of breast feeding. 
When host and environmental factors are considered conjointly as predictors of 
pediatric shigellosis, only nutritional stunting, age, and feeding mode dominate as 
risk factors. Vitamin A status was not related to the risk of shigellosis. Future 
analyses will focus on determinants of persistent diarrhea among Shigella - infected 
children. 



2M 



-s*^ 



ZOl HD 00389-04 EB 

Pro.lect Description : 

Personnel : Dr. Faruque Ahmed formerly of the Epidemiology Branch, DESPR, and now 
of Johns Hopkins University directed the data collection for this study. 
Subjects were collected while all investigators were scientists employed by the 
ICDDR.B in Bangladesh. Dr. John Clemens and Mr. Mai la Rao of the DESPR are 
CO investigators. 

Objectives; 

1. To describe the occurrence of Shigella diarrhea among Bangladeshi children 
residing in neighborhoods known to have been exposed to Shigella. 

2. To ascertain both host and environmenal determinants of Shigella diarrhea in 
these neighborhoods. 

3. To determine the frequency and determinants of Shigella diarrhea progressing 
to persistent diarrhea in these neighborhoods. 

Methods Employed : The project entailed assembly of 1934 children under 5 years 
of age, from 1335 families residing in 240 neighborhoods having a resident who 
presented for treatment of documented Shigella dysentery within 1 week of the 
initiation of active neighborhood surveillance. Following assembly of these 
children, each was followed with intensive active surveillance for one month. 
This surveillance included, at baseline, sociodemographic characterization, 
ascertainment of dietary and anthropometric status, and measurement of serum 
vitamin A. After baseline, subjects were visited according to a systematic 
schedule to ascertain symptomatic histories related to diarrhea, and to collect 
fecal specimens for microbiological characterization. 

Major Findings : Analyses of the data thus far have demonstrated that the 
occurrence of Shigella diarrhea was related to age (being highest in the third 
year of life), nutritional status (being higher in stunted children), season 
(being lowest during the monsoon season in the summer months), and breast-feeding 
status (being lower among breast-fed children, even up to 36 months of age). The 
presence of a latrine was not related to the risk, due to the fact that the 
presence of an unsanitary hanging latrine elevated the risk, while the presence 
of a sanitary slightly depressed the risk. Interestingly, the protective effect 
of breast feeding was almost entirely attributable to the increase in risk of 
shigellosis which occurred during the initial 3 months following cessation of 
breast feeding. Clinically, almost half of detected episodes were non-bloody in 
character, emphasizing the difficulties in relying on dysentery as a marker of 
Shigella infections. Isolates that were multiply antibiotic-resistant were 
associated with more swine infections. Vitamin A-status was not associated with 
disease risk or severity. 

Significance to Biomedical Research and the Program of the Institute : Pediatric 
diarrhea constitutes a major source of morbidity and mortality worldwide. 
Efforts to control pediatric diarrhea have, in the past, focused largely on 
improved case management, largely through promotion of oral rehydration therapy. 



ZOl HD 00389-04 EB 

However, these efforts have done relatively little to control the problems of 
dysentery and persistent diarrhea, for which Shigella infections are 
etiological ly of great importance and for which preventive measures may be 
necessary. Rational development of effective preventive interventions against 
Shigella requires a detailed appreciation of the epidemiology of this infection. 
This study therefore will provide information helpful both in the design of 
future interventions and in the targeting of high-risk populations for receipt 
of interventions. 

Proposed Course : During the coming fiscal year, efforts will be devoted to 
completing manuscripts on determinants of disease severity and on Vitamin A as 
a determinant of disease risk and severity. 

Publications; 



Ahmed F, Clemens J, Rao M, Banik A. Family latrines and peditric shigellosis in 
rural Bangladesh: benefit or risk? Int J Epidemiol 1994;23:856-62. 






I 



DEPARTMEhfr C3F HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SEFMCE 

NOTICE OF INTTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00392-04 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one tine between the borders.) 

Fatal Injuries to U.S. Infants, 1983-1987 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute affiliation) 

PI: R.A. Brenner Staff Fellow EB, DESPR, NICHD 

Other: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 

H.W. Berendes Director DESPR, NICHD 

A.C. Trumble Computer Specialist CSB, DESPR, NICHD 



COOPERATING UNITS (if any) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.1 



PROFESSIONAL 



0.1 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



D (b) Human tissues 



El (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The National Center for Health Statistics linked birth and death records for U.S. 
infants provides the first opportunity to assess the demographic, socioeconomic and 
prenatal risk factors associated with deaths due to injuries in infants less than 
one year of age. U.S. studies of childhood deaths have been limited to the 
extremely sparse information available on standard death certificates. Both 
descriptive and risk factor analysis of infant deaths for 1983-1988 are being 
prepared for publication. 

Of particular interest is a recent methodological analysis of infant injury deaths 
that are classified as "undetermined intent." By evaluating risk factor profiles 
of such injuries, it may be possible to classify these injuries as likely 
intentional or likely unintentional. This study has enormous public health 
implications, as such injuries could increase rates of child abuse in the less than 
one year age group by 10-15Z in the U.S. 



PHS 6040 (Rev. 5/92) 



O^^ 



ZOl HD 00392-04 EB 
Project Description 
Objectives ; 

1) To examine national fatal injury patterns among infants according to 
previously unknown risk factors, such as maternal education. 

2) To determine the degree of bias introduced by potential misclassif ication of 
injuries of undetermined intent. 

Methods Employed ; Data for this study was provided from the National Center for 
Health Statistics linked birth/infant death data sets for the years 1983 through 
1988. National, cause specific, injury death rates among infants less than one 
year of age were calculated. Univariate and multivariate analysis examined the 
effect of several matert;al sociodemographic variables (e.g. maternal education) 
and perinatal variables (e.g. birth weight) on injury outcome. 

Major Findings : The linked file data for the 1983-1988 national birth cohorts 
were pooled and analyzed. There were 6,574 deaths due to injury over this six 
year period, resulting in a death rate of 30/100,000 live births. Twenty one 
percent of deaths were classified as homicides, 75% as unintentional and 4% as 
undetermined intent. Potential risk factors ascertained from birth certificate 
information included both maternal and infant factors. Univariate analysis 
showed a significant association between each variable examined and injury death. 
Many of the characteristics were highly correlated, such as maternal age and 
marital status. 

For most of the maternal factors, relative risks for deaths classified as 
undetermined intent were the same or higher than the PR's for deaths classified 
as intentional. For example, the PR of death for infants born to mothers with 
less than twelve years of education as compared to infants born to mothers with 
at least a college education was 23.3 for deaths of undetermined intent, 7.8 for 
intentional deaths and 6.0 for unintentional deaths. Adding the deaths with 
undetermined intent to intentional deaths increased the PP in this category to 
9.0 from 7.8. Although this represents a relatively small change in relative 
risk, the absolute number of deaths now classified as intentional in the <12 
years of education category would increase from 456 to 570, a 22% increase. 

Another high risk category, with relatively small numbers of total births making 
it a practical target for interventions, is maternal age <15 years (n=61,053 from 
1983-1988 out a total of 22.6 million live births). The PR for death at ages <15 
years as compared to >25 years was 8.6 for intentional injuries, 8.7 for injuries 
of undetermined intent and 3.7 for unintentional injuries. The RR resulting from 
combining deaths of undetermined intent with intentional deaths does not change 
enough to alter the predicted number of lives saved (difference of three deaths). 
However, deaths of undetermined intent are apparently both statistically and 
logically combined with deaths due to intentional injuries in these two examples. 

Significance to Biomedical Research and the Program of the Institute : Injuries 
are the third leading cause of postneonatal death yet relatively little is known 
about the risk factors for injury death in this age group. The linked 
birth/infant death data set gives us a unique opportunity to examine risk factors 



u * 



ZOl HD 00392-04 EB 

for injury death in this age group. These results, which identify several 
maternal factors which put infants at particularly high risk of injury death 
should help public health workers in targeting preventive interventions towards 
the families of those infants who are likely to be at greatest risk. In 
additiori, preliminary rr^uits from the methodological analyses of injuries of 
undetermined intent indicate that these injuries are more likely intentional than 
unintentional in nature. These findings suggest that fatality rates from 
intentional injuries could be 10-15% greater than previously recognized. 

Proposed Course ; Initial descriptive findings were reported at the joint Society 
for Pediatric Research/American Pediatric Society/Ambulatory Pediatric 
Association meetings in 1993. Findings of the methodologic study on the 
classification of injuries of undetermined intent will be presented at the 
meeting of the American Public Health Association in October 1995. Several 
manuscripts, both descriptive and analytic, are currently being prepared. 

Publications: None at this time. 



oo 



DEPARTVENT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SERVICE 

NOTICE OF 1^^"RAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00393-04 EB 



PERIOD COVERED 



October 1, 1994 through May 31, 1995 



TITLE OF PROJECT (80 characters or less. Title must ftt on one line between the borders.) 

Trends in Death Rates from Drowning among Children, 1971-1988 



PRINCIPAL INVESTIGATOR (list other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: R.A. Brenner Staff Fellow EB, DESPR, NICHD 

Other: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Johns Hopkins University (G.Smith) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.3 



PROFESSIONAL 



0.3 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



n (b) Human tissues 



B (c) Neither 



SUMMARY OF WORK (ijse standard unreduced type. Do not exceed the space provided.) 

This project has been completed. 



■"^1 
U"-- 



PHS 6040 (Rev. 5/92) 



OEPARIWENT OF HEALTH AND HUIMAN SERVICES - PUBLIC HEALTH SEFWICE 

NOTICE OF irfTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00801-20 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit orj one line between the borders.) 

Studies Based on the Medical Birth Registries of Noirway and Sweden 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation} 

PI: A. A. Herman Visiting Scientist EB, DESPR, NICHD 



Others: K.F. Yu 

J.F. Troendle 



Mathematical Statistician 
Staff Fellow 



BMSB, DESPR, NICHD 
BMSB, DESPR, NICHD 



COOPERATING UNITS (if any) 

NIDCD (H.Hoffman); Univ. of Bergen, Norway (P.Bergsjo, L. Irgens ) ; Univ. of 
Trondheim and Natl. Inst. of Publ.Hlth., Oslo, Norway (L.Bakketeig, A.Arntzen) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
0.1 



PROFESSIONAL: 

0.1 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
n (a2) Interviews 



D (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

These studies have focused on: 1) size at birth as measured by head circumference, 
crown-heel length, and birth weight in relation to gestational age and perinatal 
mortality, 2) the tendency to repeat similar birth weight and gestational age in 
subsequent pregnancy outcomes to the same mothers, 3) perinatal mortality in 
relation to order of birth and size of sibship, 4) epidemiologic risk factors for 
preterm birth, 5) epidemiologic risk factors for small-f or-gestational age births, 
6) contribution of multiple births to perinatal mortality rates. 



PHS 6040 (Rev. 5/92) 



40 



ZOl HD 00801-20 EB 

Project Description ; 

Objectives ; These studies have been based on unique perinatal data sets, the 
Medical Birth Registries of Norway and Sweden. the objective is to compare 
United States data with that of other national population-based data sets from 
Scandinavia. These comparisons include birth weight-for-gestational age 
percentiles, birth weight-specific perinatal mortality rates and a variety of 
other sociodemographic, lifestyle, and health care comparisons. 

Methods Employed ; Analytic methods include indirect standardization of mortality 
rates by birth weight and gestational age, life-table analyses, discriminant 
analysis, examination of bivariate distributions using contour diagrams, fuzzy 
clustering of growth retarded and non growth retarded infants, non-parametric 
logistic regression, and other procedures applicable to population-based data. 
The epidemiologic approach is that of a "prospective" cohort design, which 
permits analyses by case-control, cross-sectional or longitudinal means. 

Major Findings ; 

At low birth weight the variance of last menstrual period based gestational age 
is wide and the distribution is positively skewed toward higher values. In one 
study the variance of gestational age decreases rapidly as birth weight 
increases, skewness decreases and kurtosis increases in approaching the mean of 
the birth weight distribution. Some of the wider variance and positive skewness 
of gestational age at low birth weight appears to reflect heterogeneity of 
intrauterine growth, in which infants with high values of gestational age are 
growth retarded. We show by partitioning each birth weight group into two groups 
of infants with different gestational age distributions, that at low birth 
weight, infants with low gestational ages have higher neonatal mortality rates 
but lower fetal mortality rates than infants with a higher gestational age for 
birth weight. The differences in mortality described between small infants at 
different gestational ages suggest that infants with a high LMP-based gestational 
age have experienced a slower rate of intrauterine growth. Some authors 
interpret the distributional characteristics as indications of systematic error 
in last menstrual period based assessment of gestational age. It appears from 
this study that the extent of systematic error in the estimation of LMP based 
gestational age may have been overstated in the past. The fuzzy clustering 
approach used to classify infants into lUGR groups allowed us to bypass the 
categorical approaches of lUGR classification which are prone to 
misclassif ication. Analyses of Scandinavian and United States data showed that 
among preterm infants African-American excess infant mortality increases from 
twice to sixteen times that of whites as birth weight and gestational age 
increases. Among growth retarded infants the excess mortality increases from 
twice to eight times as birth weight and gestational age decreases. 

Significance to Biomedical Research and the Program of the Institute ; The 
studies using this rich data source have provided information on several issues 
pertinent to programs of the Institute. The "repeater" studies have helped to 
elucidate some of the etiologic problems of prematurity and low birth weight. 






ZOl HD 00801-20 EB 



Studies of fetal and infant mortality, especially comparisons with available 
United States data sets, will aid understanding of the cause-specific mortality 
categories in which the United States rates may be higher than Scandinavian 
rates. The studies of lUGR classification allow for more complete examination 
of mortality and morbidity risk associated with growth abnormalities. The 
analysis of birth weight and gestational age specific mortality points to 
deficits in the care of preterm an growth retarded African-American infants. 

Proposed Course of Project : Numerous research publications have resulted from 
these collaborative efforts on the Swedish and Norwegian Medical Birth 
Registries. Further collaborations using data from both of these registries are 
continuing with Dr. Per Bergsjo, Professor, Department of Obstetrics and 
Gynecology, University of Bergen, Norway. Dr. Allen Herman continues the 
analysis of perinatal mortality risks in relation to various size at birth 
measurements using fuzzy clustering and nonparametric logistic regression 
techniques. 

Publications ; None at present. 



42 



DEPARTMEhfr C3F HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00861-13 EB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Assessment of In-Utero Fetal Growth Patterns in Relation to Outcome at Birth 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: A. A. Herman Visiting Scientist EB, DESPR, NICHD 

Mathematical Statistician BMSB, DESPR, NICHD 



Other: K.F. Yu 



COOPERATING UNITS (if any) 

NIDCD (H.Hoffman); U.Trondheim, Norway (G.Jacobsen, L.Bakketeig) ; U. Bergen, 
Norway (P.Bergsjo, T .Markestad) ; U.Uppsala, Sweden (G.Lindmark, G.Ahlstein); 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.2 



PROFESSIONAL: 

0.2 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects 
B (a1) Minors 
(a2) Interviews 



B (b) Human tissues 



n (c) Neitfier 



SUMMARY OF V\/ORK (Use standard unreduced type. Do not exceed the space provided.) 

This is a prospective study of risk factors associated with intrauterine growth 
retardation. Recruited were pregnant women before 17 weeks gestation at the 
University of Alabama in 3?irmingham and University of Trondheim, Norway (in 
collaboration with the Universities of Bergen and Uppsala) for the period of 
January 1986 through June 1988. The children are followed for five years to 
monitor cognitive, physical and behavioral development. 



PHS 6040 (Rev. 5/92) 



43 



ZOl HD 00861-13 EB 

Pro.iect Description ; 

Ob.iectives ; These studies will evaluate the predictive value of several 
obstetrical tests, including diagnostic ultrasound measurements, in assessing 
fetal growth in-utero and in relation to outcome at delivery. Follow-up 
examinations of infants will evaluate morbidity status and developmental 
progress, including several growth parameters, at one and five years of age. 

Methods Employed ; This is a population-based study conducted in Alabama and 
Scandinavia. The population in Alabama focuses on the population of Jefferson 
County, which includes Birmingham. The health care for these women is provided 
for by the county health department in affiliation with the University of Alabama 
Medical School. The Scandinavian portion of the study is being conducted in 
three university towns (Bergen and Trondheim, Norway and Uppsala, Sweden), in 
which the medical care of pregnant women in the surrounding counties is provided 
for through each of the University Hospitals. 

The goal of the study is to identify risk factors which will distinguish mothers 
having repeated smal 1-for-gestational age (SGA) births from those who have an SGA 
birth unexpected from prior pregnancy history. The study populations are 
therefore based on the selection of high-risk samples, consisting of para 1 and 
2 mothers with one or more of the following high risk characteristics; 

1. previous low birth weight delivery; 

2. previous perinatal death; 

3. serious renal disease or hypertension during pregnancy; 

4. low maternal pre-pregnancy weight (<50 kg); 

5. cigarette smoking at conception. 

In addition to the above-listed risk factors which are common to the protocols 
used at all sites, the University of Alabama protocol includes the following 
additional criteria as well: 

6. previous spontaneous abortions (2 or more); 

7. previous preterm delivery (<37 weeks); 

8. low maternal height (<157 cm); 

9. alcohol drinking during pregnancy; 

10. late onset of first prenatal care visit (26-32 weeks). 

Several statistical techniques are being used to test for significant factors 
affecting fetal growth, including multiple linear and logistic regression 
analyses. 

Major Findings ; A number of manuscripts have been written, submitted and some 
published. They deal with the relationship between a psychological profile, 
maternal size and smoking and predicting intrauterine growth retardation, 
comparison of fetal biparietal diameter, head circumference, abdominal 
circumference and femur length by race and sex, the relationship between maternal 



4-;^. 



ZOl HI) 00861-13 EB 

blood pressure, fetal growth retardation and preterm delivery; the study of 
maternal p'^ychclogical characteristics .?nd intrrty.t??rino growth retardation , as 
well as others. A special issue of Acta Obseet Gynecol will be published in 1995 
sunimari? ing findings from the first cycle of the study. The analyses of the five 
year follow op data nss been initiated. The most -tinDortant predictor of mental 
retardation at five years a>'e the social c'rcumstsnces of the family. Reduct^'on 
in mean IQ is also ;■•'■''• icted by s1i.e at birth and intrauterine growtn 
retardation. Abstracts dealing with cognitive development at 5 years were 
presented at the 3 995 Society for Gynecologic Investigation meeting and the 1995 
Society for Epidemiologic Research meetings. 

.S iM ifican ce to Biomedical Re search and the P rogr am of th e Inst itute : V a c t o r s 
which affect intrauterine growth and which may provide insight into the 
pathophysiology of intrauterine growth retardation are of considerable 
significance to the mission of this Institute. Intrauterine growth retardation 
is a significant public health prob''e(!i and associated with higher rates of 
perinatal -infant mortality and increased morbidity. 

Proposed Course of Pro .ject: The children are being assessed as they become five 
years of age. Furtrier analysis of this data are in progress. Dr. Herman 
together with scientists from both contracts will examine the intrauterine 
dynamics of fetal growl- ^mong infants with lUGR. In addition, the role of 
emerging risk factors such as psychosocial stress will be explored in these data 
sets 

C<2 nt ract Report 

The data collection prospectively during pregnancy and follow-up of infants 
through the first year of life y/as supported by the University of Alabama rn 
Birniinghaiii under NICHO contract N01-HD-4-28ii and by the University of Trondheim 
in Norway under NICHD contract NOl -HD-4"2a03. These two contracts entitled 
"Successive Small-for-Gestaticnal Age (SGA) Births: A Longitudinal Study of 
Fetal Growth and Perinatal Outcome" were initiated June 1, 1984. These two 
contracts terminated in FY 91. The data collection for the two new contracts 
entitled "roHow-up Study of Mental, Physical and Behavioral Development in Five 
Year Old Children" with the University of AlaDama in Birmingham under NICHD 
contract N01-i!D-l-3115 -■■-■•: initiated march 1, 1992 and with the University of 
Trcndheim in Norway under NICHD contact N01-HD-1-3U';7 was initiated April 1, 
1991. The FY 92 funding levels are $276,632 and $200,850, respectively. The 
current contract is devoted to the follow-up of children and their evaluation at 
five years of age. 

The FY93 funding leveis for NICHD contract N01-HD--i-3127 was $220,643. Five year 
follow-up data collection efforts were completed during FY94. 

The FY93 funding levels for NICHD contract NGl-HD-1-3116 was $291,272. Five year 
data collection efforts are expected to be complete by the end of FY94 but the 
contractor has asked for, and been granted an extension without additional costs 
to complete reporting requirements. The project is not expected to be funded in 
FY95. It was fully funded for the smont of the original contract. 



4d 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INfTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 00872-10 EB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit un one line between ttie borders.) 

Factors Associated with Premature Births; 



Missouri Follow-Back Surve-s 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: A. A. Herman Visiting Scientist EB, DESPR, NICHD 



Other: H.W. Berendes 



Director 



OD, DESPR, NICHD 



COOPERATING UNITS (if any) 

NIDCD (H.J.Hoffman); State Center for Health Statistics, Missouri Dept . of 
Health (G.Land, W.Schramm, J. Stockbauer , V.Pierson, B.Boley) 



L<\B/BRANCH 

Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.2 



PROFESSIONAL 

0.2 



OTHER: 





CHECK APPROPRIATE BOX(ES) 

B (a) Human subjects 
(a1) Minors 
(a2) Intervievi/s 



D (b) Human tissues 



n (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The objective is to obtain more accurate information relating to the very low birth 
weight (VLBW) infant, <1500 Prams, than is now available from the United States 
vital records. This objective will be accomplished by the following: 1) mailing 
or administering a questionnaire to mothers of VLBW infants, mothers of fetal 
deaths, and a sample of mothers of moderately LBW infants (1500-2499 grams) and 
normal birth weight infants (>^2500 grams) in order to obtain and verify information 
from the prenatal, perinatal, and post-neonatal periods; 2) conducting telephone 
follow-up interviews on non-respondents and incomplete respondents, and a 10 
percent sample of study mothers to obtain and/or verify information on the 
questionnaires; and 3) developing procedures for abstracting information from 
hospital and physician records, including otherwise unavailable or missing 
information on morbidity, lifestyle, and socioeconomic indicators of the study 
subjects. In addition, mortality and results of follow-up evaluations will be 
available through the first year of life for this birth cohort. Initial analyses 
of these data focused on the impact of very low birth weight and moderate low 
birth weight on cognitive development at one year, and the effect of maternal 
attitudes to weight gain on nutritional status and birth weight outcome. 



PHS 60^0 (Rev. 5/92) 



'iU' 



ZOl HD 00872-10 EB 

Pro.iect Description : 

Objectives ; This study is intended to obtain more accurate information relating 
to the very low birth weight infant, <1500 grams, than is not available. 

Methods Employed ; This is a population-based study of all very low birth weight 
(VLBW) infants and fetal deaths occurring in Missouri during a 16 month time 
period (December 1, 1989 through March 31, 1991). Moderately low birth weight 
and normal birth weight controls have been matched to the VLBW infants. The 
Missouri Department of Health has gathered data by means of questionnaires from 
mothers of infants and fetal deaths, hospitals and physicians. These data were 
linked to birth - and infant death certificates. Surviving infants were followed 
up to one year of age. The revised Denver II screening examination was used to 
evaluate cognitive development at one year. In addition the use of well-baby 
health care was assessed and illnesses during the first year of life were 
ascertained from pediatric and maternal questionnaires. 

Major Findings ; Infant mortality rates among very low birth weight infants were 
very high. The surviving very low birth weight infants had an increased frequency 
of cognitive deficits at one year. Moderately low birth weight infants had higher 
rates of developmental delay at one year when compared to normal birth weight 
infants. Women who were embarrassed about being fat gained less gestational 
weight that those who were proud of being pregnant. Those who did not worry about 
size, and those who held the baby's health of paramount importance gained more 
weight during pregnancy. 

Significance to Biomedical Research and the Program of the Institute : The study 
data will add considerably to our knowledge of risk factors and current morbidity 
conditions in the perinatal and post-perinatal periods. The initial results on 
cognitive development at one year has important implications for the impact of 
preterm birth and growth retardation on early schooling problems. 

Proposed Course of Project ; To continue the analyses of data and design a low 
term follow-up study of cognitive and physical health among preterm and growth 
retarded infants. 

Publ ications : Two abstracts on risk factors for low birth weight were published 
at the 1994 American Public Health Association meeting. 

Contract Report 

The data collection has been supported by the Missouri Department of Health under 
NICHD contract NOl-HD-6-2916. This contract entitled "Factors Associated with 
Premature Births: The Missouri Follow-back Survey" was initiated January 2, 
1986. The FY 92 funding level has been $200,000. The main objectives of this 
study are to provide new and current information about medical, sociodemographic, 
and behavioral/lifestyle risk factors of mothers who have VLBW infants and fetal 
deaths compared to those who have either moderately low birth weight or normal 
birth weight infants. The FY 94 funding for NOl-HD-6-2916 was $150,000 and 
concludes the funding for the data collection phase. 



4 



DEPARTMENT OF HEALTH AND HUIMAN SERVICES - PUBUC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02500-03 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 chamctefs or less. Title must fit on one line between the borders.) 

ETEC Seroepidemiology Evaluations in Alexandria, Egypt 



PRINCIPAL INVESTIGATOR (List other professional pe:sonret below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: J.D. Clemens Branch Chief EB, DESPR, NICHD 

Other: M.R. Rao Visiting Scientist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

NAMRU-3 Unit, Cairo, Egypt (R.Abu Elyzeed, B.Kay, L.Peruski); University of 
Alexandria (A.Mourad, M.Gaafar, M.Shasly); University of Goteborg (J.Holmgren, 
A-M. Svennerholm) . 



U\B/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.6 



PROFESSIONAL: 
0. 



CHECK APPROPRIATE BOX(ES) 

H (a) Human subjects 
(a1) Minors 
E (a2) Interview/s 



n (b) Human tissues 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Enterotoxigenic Escherichia coli (ETEC) diarrhea is hyperendemic among young 
Egyptian children, with an estimated cumulative incidence of 2.4 symptomatic ETEC 
episodes from birth to 36 months, and a balanced distribution of toxin phenotypes. 
These features make it logical to develop a field site for the evaluation of ETEC 
epidemiology and ETEC vac- lr.es in Egypt. Recently, we have begun a collaborative 
project to develop a site in Abees (near Alexandria) for the study of pediatric 
diarrhea. 

This project is following a pediatric cohort with the following aims: 1) to 
determine the age-specific incidence rate of ETEC diarrhea from birth to 35 m.onths , 
by toxin and colonization factor (CFA) phenotype; 2) to ascertain the strength of 
attribution of diarrheal symptoms to fecal isolation of ETEC; 3) to evaluate the 
protective relationship between titers of serum IgG antibodies to ETEC toxins and 
CFAs and the risk of diarrhea due to ETEC manifesting these virulence factors. 
Work began in June, 1993 on protocol design, data form development, development of 
computer systems for data entry and management, and preparation of manuals of 
procedures. In November, 1993 enrollment of subjects began. To date, approximately 
190 children have entered the study. 



PHS 6040 (Rev. 5/92) 



'ib 



ZOl HD 02500-03 EB 

Pro.iect Description ; 

Personnel ; Dr. John Clemens of the Epidemiology Branch, DESPR, is the principal 
investigator of this project, with responsibility for the epidemiological design, 
execution, and analysis of the study. Implementation of the study is 
additionally being undertaken by scientists at the NAMRU-3 Unit in Cairo (Drs. 
R.Abu Elyzeed, B.Kay, L.Peruski) and the Departments of Microbiology (Prof. 
A.Mourad) and Preventive Medicine (Prof. M. Shasly) of the University of 
Alexandria. Professors J. Holmgren and A-M. Svennerholm of the Department of 
Medical Microbiology of the University of Goteborg are consulting on 
microbiological aspects of the study. Mr. M.R. Rao of NICHD is responsible for 
the overall design of computerized data management. 

Objectives ; 

1. To determine the age-specific incidence rate of ETEC diarrhea from birth to 
35 months, by toxin and colonization factor (CFA) phenotype; 

2. To ascertain the strength of attribution of diarrheal symptoms to fecal 
isolation of ETEC; 

3. To evaluate the protective relationship between titers of serum IgG 
antibodies to ETEC toxins and CFAs and the risk of diarrhea due to ETEC 
manifesting these virulence factors. 

4. To establish, as a basis for future field trials of ETEC vaccines, the field, 
laboratory, and data management capabilities for detecting and etiological ly 
characterizing ETEC diarrhea by toxin and CFA phenotype and for evaluating 
serological responses to ETEC virulence factors. 

Methods Employed ; The study is assembling a cohort of newborns and children 
under 24 months of age, ^nd, with twice-weekly visits to homes, ascertains 
diarrheal events, which will are characterized microbiological ly for ETEC 
(including specific ETEC virulence factors) and other conventional 
enteropathogens using conventional techniques. Blood is collected by fingerstick 
every three months, and sera will be assayed for anti-ETEC IgG antibodies, for 
correlation with the ensuing incidence of diarrhea caused by ETEC exhibiting 
homologous virulence factors. 

Major Findings ; None to date (project is in progress). 

Significance to Biomedical Research and the Program of the Institute ; It has 
been estimated that among children under 5 years in the developing world nearly 
70 million episodes of diarrheal disease due to ETEC occur each year, accounting 
for approximately 700,000 deaths. Although crude, these estimates highlight the 
high disease burden of ETEC infections in the developing world, and indicate that 
a vaccine which is effective against ETEC would be of great public health benefit 
to children in these settings. Accordingly, the development and testing of 
effective vaccines against ETEC diarrhea has been identified as a specific 
priority of the Programme for Vaccine Development of the World Health 



40 



ZOl HD 02500-03 EB 

Organization. Critical to the testing of future ETEC vaccines is the development 
of field sites in which ETEC is endemic, and in which the epidemiology of ETEC 
is characterized in suitable detail for estimation of sample size requirements 
and other aspects of study design. In this regard, it is important that the 
occurrence of ETEC by toxin and CFA phenotype be known, since vaccine protection 
is expected to be homologous with respect to these virulence factors, and since 
the distribution of these factors is known to vary substantially in different 
areas. 

The present research will be conducted in Abees, Egypt, a rural, agricultural 
area (population base of approximately 20,000) located at the mouth of the Nile 
delta within 20 Km of Alexandria, Egypt's second largest city. This research 
will be conducted as a collaboration between the National Institute of Child 
Health and Human Development (NICHD) of the U.S. National Institutes of Health; 
the U.S. Navy, through the research unit in Cairo, Egypt (U.S. Navy Medical 
Research Unit No. 3: NAMRU-3); and the Faculty of Medicine, Alexandria 
University, Alexandria, Egypt (Departments of Microbiology and Community 
Medicine). The research will develop a community-based diarrheal disease study 
site that will ultimately serve as a site for evaluation of vaccines against ETEC 
and other pediatric enteric infections. In addition to providing important 
epidemiological information about childhood ETEC diarrhea, this study will 
augment the field and laooratory infrastructure of this field site, so that it 
will be suitable for the testing of future candidate vaccines against ETEC. 

Proposed Course ; This is envisioned as a four-year project. 

Publ ications; None 



uV 



DEPAHTMENT OF HEALTH AND HUMAN SSWICES - PUBLIC HEALTH SEFMCE 

NOTICE OF IhfTRAMURAL RESEAFtCH PFKDJECT 



PROJECT NUMBER 



ZOl HD 02502-03 EB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

NICHD-Health Research Board of Ireland Neural Tube Defects Study 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation} 

PI: J.L. Mills Chief, Pediatric Epidemiology Section EB , DESPR, NICHD 

EB, DESPR, NICHD 



Other: M.R. Conley Computer Specialist 



COOPERATING UNITS (if any) 

Health Research Board of Ireland and Trinity College, Ireland 



LAB/BRANCH 

Epidemiology Branch 



SECTION 

Pediatric Epidemiology Section 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.8 



PROFESSIONAL 



CHECK APPROPRIATE BOX(ES) 

S (a) Human subjects 
(al) Minors 
H {a2) Interviews 



n (b) Human tissues 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The Epidemiology Branch (DESPR) is conducting a number of studies in collaboration 
with the Health Research Board and Trinity College, Ireland. These investigations 
are designed to determine the biochemical mechanisms by which folate reduces the 
risk for neural tube defects. Data and blood samples have been collected on a 
large proportion of Irish women delivering babies in Dublin. Samples from women 
whose pregnancy ended in the delivery of a child with a neural tube defect and 
control women whose pregnancy ended in the delivery of a normal child are being 
studied. Various aspects of folate metabolism and other nutritional measures are 
being examined. The identification of vitamin B12 as a significant factor in 
preventing neural tube detects led us to examine the metabolic pathways which 
involve both B12 and folic acid. We have demonstrated that at lower levels of B12, 
women carrying a fetus with an NTD have significantly higher levels of homocysteine 
than women carrying a normal fetus. Introductory genetic studies have been very 
promising. We have identified an abnormal gene for an enzyme important to 
homocysteine metabolism in some NTD subjects and their parents. 



PHS 6040 (Rev. 5/92) 



o. 



ZOl HD 02502-03 EB 

Project Description : Dr. James Mills of the Epidemiology Branch, DESPR directs 
the NICHD-Health Research Board of Ireland Neural Tube Defects study. Subjects 
for this study have been identified in the Dublin area by our collaborators in 
the Health Research Board under the direction of Dr. Peadar Kirke. A biochemical 
investigation is being done in the laboratory of Professor John Scott at Trinity 
College, Dublin. Ms. Mary Conley of the DESPR is responsible for data center and 
Dr. Jack Lee of the DESPR is the statistician. 

Objectives : To identify the mechanism by which folate prevents neural tube 
defects, and to characterize the population at risk. 

Methods Employed : Ongoing biochemical assays are looking for differences in 
mothers who have had NTD children both during affected pregnancies and at other 
times. Genetic studies to identify markers for abnormal enzyme function are 
beginning. 

Major Findings : Having demonstrated abnormal homocysteine metabolism in mothers 
carrying neural tube defect offspring, we are pursuing the question of which of 
several enzyme pathways which could cause this abnormality is responsible. We 
have determined that a genetic abnormality in a relevant enzyme accounts for some 
(but not all) NTDs. 

Significance to Biomedical Research and the Program of the Institute : We have 
taken an important step in identifying an initial genetic component which acts 
with environmental components (folic acid and vitamin B12 levels) to produce 
neural tube defects. If it becomes possible to identify women at risk using 
markers for abnormal enzyme function, it will be possible to target vitamin 
prophylaxis. This would be of significant clinical and economic benefit. 

Proposed Course of Project : Current plans include further genetic studies in 
search of abnormalities explaining more NTD cases. We will continue to study 
mothers of NTD children during an affected pregnancy, a non-affected pregnancy, 
and in a non-pregnant state. We are developing cell culture techniques to study 
enzyme function directly. The folic acid dose and duration of therapy necessary 
to raise a sub-optimal red cell folate level to an optimal plateau is now being 
studied in the female employees of a Dublin hospital. 

Publications ; Mills JL, McPartlin JM, Kirke PN, Lee YJ, Conley MR, Weir DG, 
Scott JM. Homocysteine metabolism in pregnancies complicated by neural -tube 
defects. Lancet 1955;345:149-51. 



o;,: 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SBWICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02503-03 EB 



PERIOD COVERED 



October 1, 1994 through March 31, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

1991 Follow-up National Maternal and Infant Health Survey 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

National Center for Health Statistics (M.D. Kogan and L.A. Fingerhut] 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.2 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects 
n (a1) Minors 
B (a2) Interviews 



n (b) Human tissues 



n (c) Neither 



SUMMARY OF V^ORK (Use standard unreduced type. Do not exceed the space provided.) 

This project has been completed. 



PHS 6040 (Rev. 5/92) 



U^ 



ZOl HD 02503-03 EB 

Pro.ject Description 

Ob.iectives ; To examine the cumulative risk of injury among children from birth 
to three years old, and to provide national-level cause-specific estimates of 
non-fatal injuries for this age group. 

Methods Employed : Respondents to the maternal questionnaire of the 1991 NMIHS-LF 
were asked to describe all injuries that required a doctor's or nurse's care. 
Responses were categorized by external cause of injury. Data were analyzed using 
software for the analysis of complex multistage surveys (SUDAAN) to be 
representative of the U.S. national distribution for 1988 live births. 

Major Findings : Almost 25% of children were reported to have ever been seen by 
a medical provider for an injury. Among children who had an injury, 24.5% had 
more than one. Boys were more than twice as likely as girls to have had an 
injury. Children who received their medical care from private physicians' 
offices or HMO's and those in upper socioeconomic levels were more likely to have 
had a medically-attended injury. Falls, burns, poisoning, and being struck or 
cut were the most frequently reported cause. 

Significance to Biomedical Research and the Program of the Institute ; Injuries 
are the primary cause of death for children in the United States. Data on non- 
fatal injuries are extremely limited and non-representative. Most studies are 
based on emergency room data without a known population at risk. This study 
provides new data with demographic risk factors in a representative population- 
based U.S. sample. 

Proposed Course : Work completed. 

Publications : Kogan MD, Overpeck MD, Fingerhut LA. Medically-attended non-fatal 
injuries among preschool children: National estimates. Am J Prev Med 1995;11:99- 
104. 



b^i 



DEPARTMENT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SEFWICE 

NOTICE OF irJTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02505-03 EB 



PERIOD COVERED 



October 1, 199A through December 31, 1994 



TITLE OF PROJECT (80 characters or less. T/tle must fit on one line between the borders.) 

Infectious Disease Mortality during Infancy: United States, 1987 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: J.S. Read Senior Staff Fellow EB, DESPR, NICHD 

Other: J. Troendle Staff Fellow BMSB, DESPR, NICHD 



M.A. Klebanoff 



Research Medical Officer 



EB, DESPR, NICHD 



COOPERATING UNITS (if any) 



U\B/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 




PROFESSIONAL; 




OTHER: 


0.05 




0.05 







CHECK APPROPRIATE BOX(ES) 








D (a) Human subjects 


n 


(b) Human tissues 


(c) Neither 


D (a1) Minors 








D (a2) Interview/s 











SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 



This project is terminated. 



PHS 6040 (Rev. 5/92) 



uu 



DEPARTMENT OF HEALTH AND HUMAN SEFMCES - PUBUC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02506-03 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Lack of Age-Appropriate Immunization among Infants Born in District of Columbia 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: R. Brenner Staff Fellow EB, DESPR, NICHD 

Other: J.D. Clemens Chief EB, DESPR, NICHD 

B. Simons-Morton Health Research Specialist PRB, DESPR, NICHD 



COOPERATING UNITS (If any) 

Children's National Medical Center; D.C. Commission of Public Health; D.C. General 
Hospital; Georgetown University; Howard University; University of the District of 
Columbia 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD. NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.9 



PROFESSIONAL 

0.9 



CHECK APPROPRIATE BOX(ES) 

(a) Human subjects 
D (a1) Minors 
B (a2) Interviews 



D (b) Human tissues 



n (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

As part of the "NIH/DC Initiative to Reduce Infant Mortality" a program of research 
aimed at increasing the proportion of infants and children in the District of 
Columbia who are age-appropriately immunized has been developed. This program of 
research includes both epidemiologic and intervention (efficacy) studies. The 
phase I studies include needs assessment and feasibility studies in addition to a 
study examining determinants (as measured at birth) of immunization status at 3 and 
7 months of age. The findings from this phase will be used in the planning and 
execution of an intervention study in phase II. 

Investigators include epidemiologists, behavioral scientists, health care providers 
and administrators. The program of research was developed with input from the 
variety of perspectives represented by the investigators and the proposed studies 
have the potential to benefit from a unique blend of epidemiology, behavioral 
science, and public health and health care practice. It is our intent to develop 
interventions and identify determinants that are relevant to public health practice 
in inner cities across the US. 

This report focuses on the phase I epidemiologic studies. 



oB" 



PHS 6040 (Rev. 5/92) 



ZOl HD 02506-03 EB 

Project Description ; 

Objectives ; 1) Identify prospectively (at the time of birth) determinants of 
age-appropriate immunization among infants and children born to residents of the 
District of Columbia. 2) Assess the association between lack of age-appropriate 
immunization and other poor health outcomes. 

Methods Employed ; A birth cohort will be assembled from nursery log books. 
Baseline information will be gathered via face to face interviews with the mother 
during her hospital stay or, if this is not feasible, within two weeks of the 
del ivery. 

Demographic information as well as "consumer characteristics" (e.g. knowledge, 
attitudes, and beliefs concerning immunizations, well child care and childhood 
illness) will be collected at this time. Infants/Mothers will be contacted at 
3 months and at 7 months to assess immunization status and other health outcomes. 
Medical records will be reviewed for all health care visits. Information 
regarding health care provider characteristics and health care system 
characteristics will be gathered for all identified sites of patient care. 
(Methods for determining the current residence of infants and for retrospectively 
ascertaining immunization status and other health outcomes will also be addressed 
in the phase I studies. ) 

To determine predictors of delayed initiation of immunization, infants who are 
appropriately immunized at three months of age will be contrasted to those who 
are not. Similarly, to determine predictors of attenuation of age-appropriate 
immunization status, infants who are initially immunized at 2 months of age but 
then fail to complete the primary series in a timely fashion will be contrasted 
with infants who are appropriately immunized at 7 months. 

Major Findings : Data not yet available for analysis. 

Significance to Biomedical Research and the Program of the Institute ; 
Several factors make thi5 an extremely important project: 1) Assurance of age- 
appropriate immunization is a national public health objective. The National 
Health Promotion and Disease Prevention Objectives (Healthy People 2000) include 
a national goal of at least 90% completion of the recommended immunizations by 
24 months of age. 2) Surveys conducted in the District of Columbia in 1990 and 
1991 revealed that less than 40% of children had received all recommended 
immunizations by 2 years of age. 3) Immunization is only one of many important 
components of preventive pediatric health care for infants and children. Inter- 
ventions to improve immunization status may have the added benefit of increasing 
utilization of preventive health care, linking infants and children to systems 
that may provide continuity of care. 4) Studies in other cities have identified 
delayed receipt of the initial two month vaccine as the strongest risk factor for 
lack of age appropriate immunization at two years. This study will be one of the 
first to prospectively examine factors related to delayed initiation of 
immunization. In addition, it is desirable to identify determinants of 
immunization status prospectively (at the time of delivery) such that high risk 
families can be identified for targeted interventions. 



0/ 



ZOl HD 02506-03 EB 

Proposed Course of Project ; The hospital-based cohort study will be conducted 
over an eighteen month period. Study enrollment began in August of 1995 and will 
continue through the Spring 1996. Based on these recruitment projections, the 
3 and 7 month interviews will be completed by the Fall of 1996 and medical record 
reviews will be completed early in 1997. 

Publications ; None yet. 






DEPAPTTMEfiT OF HEALTH AND HUIUIAN SEFMCES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02507-03 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Prostaglandin Excretion in Preeclampsia 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: 
Other: 



J.L. Mills 
R.J. Levine 
J.D. Clemens 



Chief, PES 
Expert 
Chief. EB 



EB, DESPR, NICHD 
EB, DESPR, NICHD 
EB, DESPR, NICHD 



COOPERATING UNITS (if any) 



L^B/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.2 



PROFESSIONAL 
0.2 



OTHER: 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
n (a2) Interviews 



(b) HurTian tissues 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

A disturbance of prostaglandin metabolism is currently believed to be one of the 
major mechanisms in the pathogenesis of preeclampsia. Most previous studies of 
this theory, however, have included only women who had already developed clinically 
overt disease. In order to investigate whether prostaglandin abnormalities precede 
the onset of preeclampsia, Te have designed a nested case-control study using 
stored urine specimens prospectively collected as early as thirteen weeks gestation 
from women participating in the NICHD Trial of Calcium to Prevent Preeclampsia 
(CPEP). In this study, we will compare the urinary excretion of prostaglandin and 
thromboxane in preeclampsic women before the diagnosis of preeclampsia to that in 
normal pregnant women at comparable gestational ages. Because half of the women in 
the CPEP trial will be taking two grams of oral calcium daily, we will also be able 
to examine the effect of oral calcium supplementation on the excretion of these 
prostaglandin metabolites. 



PHS 6040 (Rev. 5/92) 






ZOl HD 02507-03 EB 

Project Description : Dr. James Mills, Dr. Richard Levine, and Dr. John Clemens 
are collaborating on this project. The laboratory analyses will be performed by 
Dr. Jackson Roberts at Vanderbilt University. 

Objectives : To determine whether an imbalance of prostaglandin metabolism 
precedes the clinical onset of preeclampsia. 

Methods Employed : Vanderbilt University is assaying specimens (blinded to 
status) from women who developed preeclampsia and normal control women. Thus 
far, nearly 200 urine specimens have been assayed for the prostacyclin metabolite 
2,3-dinor-6-keto-PGFi aip,,a and the thromboxane metabolite ll-dehydro-TxB2. 

Major Findings : None yet. 

Significance to Biomedical Research and the Program of the Institute : 
Preeclampsia occurs in 5-10% of all pregnancies, and causes substantial maternal 
and neonatal morbidity. Determining the pathogenic mechanisms underlying this 
disease will assist in development of rational prediction tests and therapies. 

Proposed Course : Samples will continue to be analyzed until the CPEP trial is 
completed. Then the code will be broken and the final analysis will be 
performed. 

Publications: None 



DEPARTMEI^ OF HEALTH AND HUMAN SEFNICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02510-03 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Diet, Maternal Nutritional Status, Blood Pressure and Fetal Growth 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiitallon) 

PI: N. Tafari Visiting Scientist EB, DESPR, NICHD 



Other: J.D. Clemens 
H.¥. Berendes 
Y. Johnson 



Chief 
Director 
IRTA Fellow 



EB, DESPR, NICHD 
DESPR, NICHD 
EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Ethiopian Nutrition Institute (Z.W. Gabriel, E. Woohib) 



U\B/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.1 



PROFESSIONAL: 
0.1 



OTHER: 




CHECK APPROPRIATE BOX(ES) 

E (a) Human subjects 
n (a1) Minors 
(a2) Interviews 



n (b) Human tissues 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The study was sponsored by the City Council of Addis Ababa as part of health and 
nutritional assessment of low income families prior to planning a food 
supplementation program for women and children. This particular program was 
initiated because of sudden increase in the price of essential food items following 
implementation of government policies that preferred state control of production 
and distribution of all essential commodities. 

The study of the effect of home diet on fetal growth arose from the observation 
that in Ethiopia, where severe deficiency in food energy is prevalent, there 
appears to be no corresponding deficit in fetal growth. This observation is at 
variance with the generally held view that dietary energy deficit during pregnancy 
leads to fetal growth retf-rdation. However, several lines of recent evidence 
suggest that fetal growth is protected through adaptive processes. Among possible 
adaptive mechanisms area: 1) higher physical work efficiency; 2) greater 
mobilization of maternal energy reserves (fat deposits); and 3) hemodynamic 
adjustments to maintain adequate uterine perfusion. 

Data analysis to date showed that the dietary energy deficits in low income 
families were associated with deficiencies in nutritional status of the gravida 
during the third trimester and the puerperium as reflected by reduced skinfold 
thickness and body mass index. Third trimester diastolic blood pressure was 
significantly reduced in poorly nourished gravida. Although a statistically 
significant difference of 180 g in birth weight was detected between the offspring 
of the undernourished and adequately nourished, it is not yet clear whether this 
difference is due to diet alone. 



PHS 6040 (Rev. 5/92) 



G... 



ZOl HD 02510-03 EB 

Project Description 

Ob.iectives ; The objective of the project is to investigate the effect of home 
diet on fetal growth in a population with long term energy undernutrition. 

Methods Employed : The study was part of a health and nutrition survey of low 
income families in Addis Ababa during 1987-88 when it was felt that food 
availability at the level of the household was reduced as a result of disturbance 
of the market forces by government policies that preferred state control of 
production and distribution of all essential commodities. The study was 
conducted in four community-based primary health care facilities serving the 
poor, and in one privately financed prenatal health service serving the affluent. 
Dietary energy, protein and selected nutrient intake were estimated from 4- 
consecutive-day weighed measurements of the home diet. Individual food items 
were weighed before cooking. The meal is weighed again before consumption. The 
daily household consumption was estimated from the difference in weight between 
food prepared and left over food. The nutrient content of the home diet was 
estimated using values of the "National Food Composition" tables. Household 
energy intake was expressed as multiple of basal metabolic rate (BMR). The 
"household basal metabolic rate" (HBMR) was the sum of the individual member's 
BMR and was estimated from published tables (WHO, Tech Report Series 724, 1985). 
According to WHO the survival energy requirement that allows only for the minimum 
of activities is 1.27 times BMR. Nutritional status of the pregnant woman and 
her family was assessed using selected anthropometric measurements, including 
weight, height and skinfold thickness at selected sites. Other variables 
considered include weight, skinfold thickness and blood pressure changes during 
pregnancy and puerperium. 

Major Findings ; Preliminary data analysis on women who participated in the home 
diet survey is completed. Women attending community-based prenatal clinics had 
lower socioeconomic profile than women using private prenatal facilities as 
reflected by lower income and low food energy intake. The poor gravida also 
tended to be younger with higher parity. The low energy intake in these women 
was reflected in their poor nutritional status -- low body mass index and reduced 
skinfold thickness. When the gravida were stratified into those with household 
energy intake to expenditure ratio <1.27 and >1.27 the women with lower energy 
intake to expenditure ratio had a significantly lower diastolic blood pressure 
and the mean birth weights of their newborns was 180 g lighter. 

As anticipated, the deficit in birth weight is smaller than the actual deficit 
in food energy intake and the degree maternal undernutrition as assessed by 
anthropometric measurements. Data exploration and analysis on the entire cohort 
is in progress in an attempt to identify adaptive mechanisms that tend to 
minimize the effect of prenatal food energy deficiency on fetal growth. 

Significance to Biomedical Research and the Program of the Institute ; This 
cohort offers a unique opportunity to study the effect of maternal food energy 
deficit to near starvation level, the resulting nutritional status and blood 
pressure changes during the second half of pregnancy on fetal growth. If the 
study yields information on adaptive mechanisms protecting fetal growth, such 
knowledge may help health planners to target pregnant women who lack these 



B;- 



ZOl HD 02510-03 EB 

adaptive traits. Since the women were followed through delivery to the end of 
the puerperium it can also provide information on the additional effect of 
pregnancy on postnatal nutritional status of undernourished women. 

Proposed Course of Project ; Data analysis and manuscript preparation will 
continue during the next fiscal year. 

Publ ications ; None yet. 



63 



DEPAFrrMEhfT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SERVlCe 

NOTICE OF im"RAMURAL RESEAFtCH PROJECT 



PROJECT NUMBER 



ZOl HD 02511-02 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (30 characters or less. Title must fit on one line between the borders.) 

Immunogenicity of Routine Childhood Vaccines in HIV Positive Children 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: J.S. Read Senior Staff Fellow EB, DESPR, NICHD 

Other: J.D. Clemens Branch Chief EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

FDA (B.Anthony); NERI (D.Brambilla ) ; NIAID (M.G. Fowler ) ; Columbia University 
(J.Pitt); University of Illinois (K.Rich) 



LAB/BRANCH 



Epidemiology Branch 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.25 



PROFESSIONAL: 

0.25 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
□ (a2) Interviews 



B (b) Human tissues 



D (c) Neittier 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The immunogenicity of routine childhood vaccines in HIV-infected children is not 
well understood and may be substantially less than in non- infected children. It is 
important to determine the immunogenicity of such vaccines in this population and 
thus identify the most immunogenic vaccines and vaccine schedules in this 
population. Data from the Women and Infants Transmission Study (WITS) will be 
analyzed. 



PHS 5040 (Rev. 5/92) 



»-i /.: 



ZOl HD 02511-02 EB 

Project Description ; 

Objectives ; This study was designed to address the following hypotheses; 1) The 
magnitude and duration of the immune response of HIV-infected children to these 
vaccines will be less than that of noninfected children; 2) Among HIV-infected 
children, the serologic response to these vaccines will be inversely related to 
the severity of HIV-related symptoms, i.e., HIV-infected children who are 
clinically symptomatic at the time of vaccination will respond less well than 
asymptomatic HIV-infected children; and 3) Among HIV-infected children there may 
be clinically significant differences in the magnitude and duration of the immune 
responses to the vaccines. 

Methods Employed ; Children of HIV-infected women enrolled in the Women and 
Infants Transmission Study (WITS) (N=443 as of March 1, 1993) will be eligible 
for inclusion in this study. All children remain under surveillance until 36 
months of age, during which time all routine childhood immunizations are 
administered and venipuncture is performed on a regular basis. Data regarding 
types of immunizations received, dates of immunizations, and vaccine 
manufacturers are stored in the WITS database. 

Major Findings ; Primary analyses will compare magnitude and duration of the 
immune responses of HIV-infected children to DTP and Hib vaccines to those of 
noninfected children. Further analyses will compare the serologic responses to 
these vaccines in symptomatic and asymptomatic HIV-infected children. 

Significance to Biomedical Research and the Program of the Institute ; HIV 
infection has become the most common cause of immunodeficiency in children in 
many parts of the world. In the U.S., the number of individuals in the pediatric 
age group infected with rilV is rapidly increasing. Vaccine-preventable diseases 
continue to cause significant morbidity and mortality in HIV-infected children 
in the U.S. Because HIV-infected children will remain at risk for vaccine- 
preventable diseases until these diseases are eradicated, it is important to 
determine the immunogenicity of routine childhood vaccines in this population 
and, eventually, to identify the most immunogenic vaccines and vaccine 
administration schedules in this population. 

Proposed Course of Project : The serological testing is anticipated to be 
completed within the next fiscal year. 

Publications ; None yet. 



{Jb 



DGPAmMEhfT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SERVICE 

NOTICE OF irJTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02512-02 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Birth Certificate Linkage to Growth and Health Measures using NHANESIII 



PRINCIPAL INVESTIGATOR (List other professional personnei below the Principal Investigator.) (Name, title, laboratory, and institute afTiliation) 

PI: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

National Center for Health Statistics (R.Kuczmarski, G.Gay, J.Findlay) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.1 



PROFESSIONAL: 

0.1 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
D {a2) Interview/s 



n (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The third National Health and Nutrition Examination Survey (NHANESIII) has 
collected descriptive data, physical measures, and biomedical samples (blood and 
urine) on a representative U.S. sample of children at ages two months through six 
years with oversampling for blacks and Mexican Americans. Linkage of the extensive 
data from NHANESIII to the c'lild's birth certificate to use the prenatal and birth 
information has begun. All of the states were sent requests for the relevant birth 
certificates with the signed permission of parents of the examined child. The data 
from the first three states have been received. These data are being used to test 
the computerized matching of NHANESIII and birth certificate data. 

Results of the linkage will support research efforts to do the following: 1) To 
determine if low birth weight or premature children have subsequent poor health as 
measured by biomedical markers in a representative national sample of children 
through age six; 2) to assess the effects of ethnic/racial and socioeconomic 
factors on these outcomes; 3) to develop normative growth and other biomedical 
scales for a U.S. representative population, and black and Mexican-American 
children, according to a risk profile at birth; and 4) to develop comparisons 
between maternal and proxy reports of children's risk factors at birth and actual 
measurements for use in other studies. 



T> 



PHS 6040 (Rev. 5/92) 



V.' 



ZOl HD 02512-02 EB 

Project Description ; 

Ob.iectives : 1) To determine if low birthweight or premature children have 
subsequent poor health as measured by biomedical markers in a representative 
national sample of children through age six; 2) to assess the effects of 
ethnic/racial and socioeconomic factors on these outcomes; 3) to develop 
normative growth and other biomedical scales for a U.S. representative 
population, and black and Mexican-American children, according to a risk profile 
at birth; and 4) to develop comparisons between maternal and proxy reports of 
children's risk factors at birth and actual measurements for use in other 
studies. Major outcomes of interest will include 1) weight and height growth 
scales by risk profiles at birth; 2) effects of passive smoke exposure in the 
household controlling for prenatal factors; and 3) other health measures 
collected both by questionnaire and biomedical measurement. 

Methods Employed ; The third National Health and Nutrition Examination Survey 
(NHANES III) has collected descriptive data, physical measures, and biomedical 
samples on approximately 9,000 children at ages two months through six years with 
oversampling for blacks and Mexican Americans. The extensive data from NHANES 
III will be linked to the child's birth certificate to use the prenatal and birth 
information. NCHS is obtaining the certificates from the States and linking the 
data. Data analysis will require application of appropriate software for 
analysis of the complex multistage sample design of NHANES III for both 
descriptive and multivariate findings. 

Major Findings ; None available yet. 

Significance to Biomedical Research and the Program of the Institute ; Our 
ability to determine the health outcome of children classified as high risk at 
birth compared to other children has been extremely limited. Children who have 
been followed are seldom representative of even small areas. Normative 
comparisons for hypothesis testing of poor health outcomes have been based on 
scales developed from small, potentially biased populations. Use of this 
nationally representative sample including growth and other measures is a unique 
opportunity to provide norms for both the clinical and research community. 

Proposed Course of Project ; After NCHS obtains and links birth certificates, we 
will perform both descriptive and multivariate predictive analysis for 
publication in peer-reviewed journals. Other research using the blood and urine 
samples has been proposed. 

Publications; None to date. 



6v 



DEPARTMENT OF HEALTH AND HUMAN SERWCES - PUBLIC HEALTH SERVICE 

NOTICE OF im"RAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02513-02 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

High Status and Risk Factors Analysis of Premature High-Risk Deliveries 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Pnncipal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 



Other: A.C. Trumble 



Chief, Computer Sciences Branch CSB, DESPR, NICHD 



COOPERATING UNITS (if any) 

March of Dimes (J.Petrini, K.Damus) 



LAB/BRANCH 

Epidemiology Branch 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.05 



PROFESSIONAL 
0.05 



OTHER: 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
n (a2) Interviews 



G (b) Human tissues 



B (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Trends toward increasing survival of infants born at less than 750 grams (about 1- 
1/2 pounds) have suggested higher prevalence of complications related to birth and 
continuing health deficits. Dr. Overpeck documented the status for the births, 
subsequent deaths, and survival in the U.S. birth cohort of 1983. She has provided 
background and technological support for staff of the March of Dimes (Joann 
Petrini) to complete the collaborative analysis to assess changes in survival and 
mortality of these extremely low birth weight infants. The analysis will compare 
the baseline years before introduction of surfactant therapy (1983), years 
incorporating the experimental phase of surfactant therapy (1987-88), and the 
latest years of available data which include births associated with both surfactant 
and antenatal corticosteroid therapy (1990-92). Deaths to infants born in the 
baseline years at weights less than 750 grams (about 24 weeks gestation) 
contributed about 25 percent of total infant mortality in 1983. Over 40 percent 
of deaths prior to six days of life were from these smallest least mature infants. 



'SB 



PHS 6040 (Rev. 5/92) 



I 



ZOl HD 02513-02 EB 

Pro.ject Description 

Objectives ; To assess changes in survival and mortality of live-born infants 
weighing less than 750 grams in the U.S. 

Methods Employed ; Summary data prepared by NCHS for release with the public use 
tapes of the 1983 Birth Cohort Linked Birth/Infant Data Set were analyzed for 
U.S. resident deaths of infants born in 1983. The latest data available from 
this source (probably 1987-89) is being compared for the total U.S. and local 
areas. Multivariate risk factor analysis will be completed. 

Major Findings : Deaths in infants born at less than 500 grams (about 20 weeks 
gestation) contributed about 10 percent of total infant mortality in 1983. 
Deaths to infants born at less than 750 grams contributed about 25 percent. 
About 33 percent of all Black deaths occur to infants born at less than 750 
grams; 22 percent of White deaths occur in this birth weight range. Over 40 
percent of deaths prior to six days of life are from these smallest, least mature 
infants. 

Significance to Biomedical Research and the Program of the Institute ; The 
analysis of birth weight-specific mortality rates focuses attention on the timing 
of interventions needed to reduce infant mortality and to prevent such high risk 
deliveries. Birth weight-specific comparisons at the national and local levels 
will be made to assess differences in risk factors and outcomes. The time 
periods analyzed compare years encompassing the phases of testing and use of both 
surfactant and antenatal corticosteroid therapy. 

Proposed Course ; Changes in survival rates since 1983 will be analyzed with 
multivariate risk factor assessment. Publication in peer-reviewed journals and 
dissemination of local data through the March of Dimes is expected. 

Publications: None to date. 



yU 



DEPARTMENT OF HEALTH AND HUMAN SERMCES - PUBUC HEALTH SERVICE 

NOTICE OF im"RAMURAL RESEAFICH PROJECT 



PROJECT NUMBER 



ZOl HD 02514-02 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (SO characters or less. Title must fit on one line between the borders.) 

Childhood Drowning Deaths - A National Analysis of Death Certificate Information 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal investigator.) (Name, title, laboratory, and institute affiliation) 

PI: R.A. Brenner Staff Fellow EB, DESPR, NICHD 

Other: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Johns Hopkins University (G.Smith); Consumer Product Safety Commission 
(A. McDonald) 



LAB/BRANCH 



Epidemiology Branch 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS; 



0.2 



PROFESSIONAL; 



0.2 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
n (a2) Interviews 



n (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not jxceed the space provided.) 

Working in collaboration with the Consumer Product Safety Commission (CPSC) 
information is being abstracted from death certificates for all non-boat related 
drowning deaths in children and adolescents less than 20 years of age. The free 
text portion of the death certificate often includes important information that is 
not included in national mortality files. Of particular importance is the specific 
site of the occurrence (e.g. swimming pool, lake, irrigation ditch, etc.) as 
interventions to prevent drowning deaths are site specific. Results from this 
study will provide the first in depth look at the circiamstances surrounding 
drowning deaths at a national level. 



PHS 6040 (Rev. 5/92) 



iV- 



f 



ZOl HD 02514-02 EB 

Pro.ject Description ; 

Gb.iectives : To describe the epidemiology of drowning in the U.S. through the 
abstraction and analysis of death certificate information. 

Methods Employed ; Through an agreement with the Consumer Product Safety 
Commission, death certificates will be purchased from the fifty states and two 
major health jurisdictions (New York City and Washington, D.C.) for deaths in 
which the underlying cause of death is a nonboat-related drowning (E-code 910.0- 
910.9, 954, 964 and 984) and the age of the victim at the time of death is less 
than 20 years. Information from the purchased death certificates will be 
abstracted and coded to provide a uniform dataset for analyses of drowning 
deaths. 

Data will be used in descriptive analyses of the circumstances of drowning, with 
particular focus on stratii^ied analyses of the specific site of drowning by age 
at death and state of occurrence. Other variables of interest include sex, race, 
products associated with the death, and other details surrounding the event (e.g. 
the involvement of alcohol) abstracted from the narrative portion of the death 
certificate . 

Major Findings ; Results not yet available, however, a recent review of the 
literature and the epidemiology of drowning in the adolescent age group has been 
completed by two of the investigators on this study. The authors again 
identified the lack of data at the national level on the primary circumstances 
surrounding drowning deaths with over 70% of drownings in the adolescent age 
group being coded to the "other" or "unspecified" categories in national 
datasets. 

Significance to Biomedical Research and the Program of the Institute ; In the 
United States, drowning is the second most common cause of unintentional injury 
death among children and adolescents. In the one to two year old age group it 
is the single leading cause of injury death. Other groups at increased risk of 
drowning include adolescent males (particularly black adolescent males) and, in 
recent years, infants less than one year of age. 

Interventions to prevent drowning deaths are dependent not only on the age of the 
victim but also on the specific location and circumstances surrounding the event. 
Unfortunately, national mortality data often do not provide sufficient 
information regarding the circumstances of these drowning deaths to allow injury 
prevention experts to design appropriately targeted interventions. In particular 
the specific site of the drowning (i.e. swimming pool, lake, bucket, irrigation 
ditch etc.) can not be identified from computerized mortality data because the 
E-Code (the ICD code for external injuries) is not specific for site of drowning. 
The hard copy of death certificates are a valuable source of information on the 
circumstances surrounding drowning deaths, including the specific site of the 
event. 

This study will be the first to describe, at a national level, the specific 
circumstances surrounding drowning deaths thus enabling the development of 
appropriately targeted interventions. 



1 -! 



ZOl HD 02514-02 EB 

Proposed Course of Pro.iect ; As outlined in the interagency agreement with CPSC, 
death certificates for all drowning deaths received since January 1, 1994 have 
been retained by CPSC. CPSC has coded information from those drowning deaths 
that were not product related. (Coding of information from product related 
deaths is in the usual scope of work at CPSC.) Contr'acts with the 50 states and 
two major health jurisdictions (New York City and Washington, DC) for the 
purchase of death certificates have been modified to include all non-boat related 
drowning deaths as outlined in the interagency agreement. These modifications 
took effect in FY'95. As of June 1995, 998 death certificates were abstracted 
for deaths occurring in 1993 and 576 for 1994. Under the terms of the agreement, 
CPSC will continue coding this death certificate information through the end of 
FY'96. Due to delays in reporting (from the states to CPSC) it is anticipated 
that the most complete dataset will be compiled for the 1994 calendar year. 

Publications ; 

Smith G, Brenner R. The changing risks of drowning for adolescents in the U.S. 
and effective control strategies. Adoles Med: State-of-the-Art-Reviews 
1995;6:153-69. 



( (Z 



DEPAFnWEhfT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SEFMCE 

NOTICE OF INTTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02515-02 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.} 

Prevention of Childhood Injuries 



Phase I Injury Surveillance 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: R.A. Brenner Staff Fellow EB, DESPR, NICHD 

Other: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Children's National Medical Center (P. Scheldt); D.C. Commission of Public Health; 
D.C. General Hospital; Georgetown University; Howard University; University of the 
District of Columbia 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.5 



PROFESSIONAL: 



0.5 



CHECK APPROPRIATE BOX(ES) 

H (a) Human subjects 
D (a1) Minors 
(a2) Interviews 



n (b) Human tissues 



D (c) Neither 



SUMMARY OF WORK (tjse standard unreduced type. Do not exceed the space provided.) 

As one component of the "NIH/DC Initiative to Reduce Infant Mortality" a program of 
research addressing the threat of injuries and neglect to infants and toddlers in 
the District of Columbia has been developed. The study is divided into two phases. 
During phone one a comprehensive, city-wide injury surveillance system will be 
established. This surveillance will include all injuries in the target age group 
(birth through two years) that result in an emergency room visit, hospitalization 
or death. Information will also be obtained from the Department of Family Services 
on substantiated cases of abuse and neglect. 

The information obtained in phase I will guide the design of specific targeted 
interventions, for the pr-:-'>'-cntion of injuries, that will be implemented and 
evaluated in phase II. A randomized intervention trial of local community entities 
(neighborhoods or wards) is proposed in which epidemiologically based environmental 
interventions are applied to one group, behaviorally based interventions to prevent 
abuse and neglect to a second group and epidemiologic surveillance alone to a third 
group. As an essential component of injury prevention, injury surveillance system 
will continue for all three groups, throughout phase II. 

The remainder of this report focuses on the phase I surveillance activities as 
detailed plans for the intervention phase are dependent on the results of the 
surveillance and thus have not yet been developed. 



PHS 6040 (Rev. 5/92) 



( KJ 



ZOl HD 02515-02 EB 

Pro.ject Description ; 

Objectives : 1) To collect citywide epidemiological data to define causes and 
patterns of injuries, both intentional and unintentional, that will enable 
investigators to develop effective intervention approaches in phase II of this 
study. 2) To determine the direct costs of intentional and unintentional 
injuries in the target population. 

Methods Employed ; Information will be abstracted from medical records and 
billing records of children ages 0-2 years who were seen in an emergency room or 
admitted to a hospital for an injury. All non-military hospitals in which 
children from the District of Columbia routinely seek care will be included in 
the surveillance. In addition, at three study sites a questionnaire will be 
administered to the parent or caretaker at the time of the ER visit or 
hospitalization. This questionnaire includes information on circumstances 
surrounding the injury event and other potential risk factors. Intake records 
from the Department of Family Services will be abstracted to provide information 
on cases of substantiated child abuse and neglect. 

The leading causes of fatal and nonfatal injuries will be identified. Emergency 
room injury incidence rates, hospital admission rates and fatal injury incidence 
rates will be calculated. Injury rates will be compared between census tracts 
to identify communities in which children are at increased risk. Where community 
data exists on sociodemographic characteristics (e.g. maternal age) analyses will 
compare the characteristics of the injured population to those of the community 
to identify characteristics which place various subgroups at increased risk of 
injury. 

Major Findings ; Data not yet available for analysis. 

Significance to Biomedical Research and the Program of the Institute : Injuries 
are the third leading cause of postneonatal death and the leading cause of death 
after the first year of life. Studies have shown that many of these injuries are 
preventable. Another serious problem is that of child neglect. In 1992, the 
D.C. Commission of Social Services investigated 4,886 families (with a total of 
10,922 children and infants) for neglect. Approximately 25% of reported cases 
involve children less than three year of age. 

The "NIH/DC Initiative to Reduce Infant Mortality" is aimed at addressing 
multiple causes and factors related to the high infant mortality rate in the 
District of Columbia. Among the types of research called for by the initiative 
is the development of interventions to prevent or reduce injuries. If 
interventions in phase II are effective, this program of research will not only 
reduce injuries among infants and young children of the District of Columbia but 
will also serve as a model program for other inner city communities. 



'V 



ZOl HD 02515-02 EB 

Proposed Course of Pro.ject ; Data collection for the Phase I study will begin in 
September of 1995 and continue through September of 1996. A detailed protocol 
of the Phase II intervention study will be developed based on the results of the 
initial six months of injury surveillance data. However, due to expected 
seasonal variations in the incidence and causes of injury, the protocol will not 
be finalized until a complete year of data are collected and analyzed. 

Publications: None to date. 



to 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBUC HEALTH SERVlCe 

NOTICE OF im"RAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02516-02 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 



The Role of Bathtub Rings and Seats in Infant Drowning Deaths 



PRINCIPAL INVESTIGATOR (List other professional personnel below [he Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: R.A. Brenner Staff Fellow EB , DESPR, NICHD 



COOPERATING UNITS (if any) 

Consumer Product Safety Commission (R.Rauchschwalbe) ; John Hopkins University 
(G.Smith) 



LAB/BRANCH 

Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.2 



PROFESSIONAL: 



0.2 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
n (a2) Interview/s 



D (b) Human tissues 



B (c) Neither 



SUMMARY OF V^/ORK (Use standard unreduced type. Do not exceed the space provided.) 

Drowning is a leading cause of injury death among children in the United States. 
Recent studies have shown that, unlike other age groups, rates of death from 
drowning are actually increasing among infants. Approximately hOZ of infant 
drownings occur in the bathtub. In this study we examine the involvement of bath 
rings and bath seats (products intended to support an infant in the sitting 
position during bathing) in drownings among infants and toddlers in the United 
States. 

Using data from the Consumer Product Safety Commission, 21 drowning deaths, in 
which a bath seat or bath ring was in use at the time of submersion, were 
identified. Infants ranged in age from 5-15 months with a median age of 8 months. 
In a series of focus group discussions, parents reported that they were more likely 
to leave a child unattended momentarily in the bathtub if the infant was contained 
in a bath seat or ring. There was a reported lapse in adult supervision in 19 of 
21 (902) of the incidents. This study is the first in the medical literature 
alerting health professionals to the potential hazards of these products and 
reinforces the need to counsel parents on the dangers of leaving small children and 
infants unattended in the bathtub. 



PHS 6040 (Rev. 5/92) 



/o 



ZOl HD 02516-02 EB 



Pro.ject Description ; 



Ob.iectives : To describe drowning and near drowning incidents involving the use 
of bathtub rings or seats. 

Methods Employed ; Data were obtained from the Consumer Product Safety Commission 
(CPSC) on deaths and near drownings involving bathtub seats/rings. Incidents 
were reported to CPSC through a number of sources including: both a voluntary and 
paid Medical Examiner's and Coroner's Alert Program; reports from a randomly 
selected number of hospitals that report all product-related emergency room 
injuries (NEISS); death certificates for product related incidents which are sent 
in from each of the fifty states and two major health jurisdictions (New York 
City and Washington, DC); a newspaper clipping service and a toll-free 800 line. 
Bathtub seat/ring incidents identified by CPSC Epidemiology staff are referred 
to the Division of Corrective Actions for in-depth investigation (IDI) 
assignment. 

Reported incidents that occurred on or before December 31, 1994 and for which the 
IDI was completed by July 31, 1995 are included in this study. To be eligible 
for inclusion in this case series a bathtub seat had to be in use at the time of 
death or injury and the primary cause of death or injury had to be due to 
drowning or near drowning. Major findings are summarized below. 

Major Findings ; 

1. Twenty-one deaths and six near drownings were identified. The first incidence 
occurred in August of 1983 and the most recent occurred in October of 1994. 

2. The age at death ranged from 6 to 15 months, with a median age of 8 months. 

3. There was generally a history of a lapse in adult supervision (either leaving 
the infant alone in the tub or in the care of an older child.) Among the fatal 
incidents, the lapse in adult supervision ranged from 0-35 minutes with a median 
of 5 minutes and a mean of 6.4 minutes. 

4. Results from three separate focus panels showed that there is a false sense 
of security with bath rings and seats in that mothers/caregivers are more likely 
to feel comfortable leaving the infant alone in the tub for a few minutes when 
these products are in use. 

Significance to Biomedical Research and the Program of the Institute ; Drowning 
is an important preventable cause of death in childhood. Among infants and 
toddlers a large number of these drownings deaths occur in the bathtub. This 
study is the first in the medical literature to identify the involvement of bath 
rings and bath seats in drownings among infants and toddlers. It is hoped that, 
once alerted to the problem, health care professionals will warn parents and 
caregivers about the potential hazards of these products. Additionally it is 
thought that there is currently substantial underreporting of incidents involving 
these products. It is anticipated that after results from this study are 
published reporting of events (particularly non fatal events) involving these 
products will increase giving a more accurate estimate of the extent of the 
problem. 



7 



ZOl HD 02516-02 EB 

Proposed Course of Pro.ject : Preliminary findings were reported at the combined 
Society for Pediatric Research/American Pediatric Society/Ambulatory Pediatric 
Association meetings in May of 1995. A manuscript is currently being prepared 
and will be submitted for peer review. 

Pub! ications; None to date. 






DEPARTMEhrr OF HEALTH AND HUkMN SSWICES - PUBLIC HEALTH SEPMCE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02517-02 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title rriust fit on one line between the borders.) 

First Week Changes in Birthweight and Other Neonatal Anthropometric Parameters 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: N. Tafari Visiting Scientist EB, DESPR, NICHD 



Other: J.D. Clemens 
M.R. Rao 
J.F. Troendle 



Chief 

Visiting Scientist 

Staff Fellow 



EB, DESPR, NICHD 
EB. DESPR, NICHD 
BMSB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Department of Pediatrics, Addis Ababa University, Addis Ababa, Ethiopia 



LAS/BRANCH 

Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.30 



PROFESSIONAL 

0.30 



CHECK APPROPRIATE BOX(ES) 

E (a) Human subjects 
B (a1) Minors 
B (a2) Interviews 



D (b) Human tissues 



D (o) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Classification of newborns according to birthweight and gestational age is a useful 
clinical and epidemiologic tool to identify newborn infants at high risk for excess 
morbidity and mortality. In developing countries birthweight and gestational age 
cannot be measured directly. Moreover, preterm and low birth weight lose up to lOZ 
of their birthweight during the first week. Using the statistical technique of 
recursive partitioning we recently demonstrated that head and chest circumference 
measurements accurately i'^entif ied preterm, low birthweight newborns on data from 
Ethiopia. The rationale for employing other body measurements as surrogate for 
birthweight is dependent on their stability during the first week. We have also 
examined the results of serial measurements of head, chest, midarm circumference, 
body weight and length measurements in a cohort of 600 Ethiopian newborns in order 
to determine their pattern during the first week. 



PHS 6040 (Rev. 5/92) 



ZOl HD 02517-02 EB 

Pro.ject Description : 

Objectives ; To illustrate that measurements projected to serve as surrogates for 
birthweight such as head and chest circumferences are stable over the first week 
of life and have the same sensitivity and specificity in identifying preterm, low 
birthweight newborns. 

Methods Employed ; Body weight and length and, head, chest and midarm, were 
obtained on days 1, 3 and 7 on 300 infants with known gestational ages. Repeated 
measures of analysis of variance and recursive partitioning would be used to test 
the stability of surrogate measurements in predicting birthweight and gestational 

age. 

Major Findings ; Head, chest and midarm circumferences were stable while 
significant decline in birthweight was apparent. 

Significance to Biomedical Research and the Program of the Institute : Effects 
of diverse health experiences of the mother prior to and during pregnancy are 
accurately reflected by birth weight and the duration of gestation. 
Identification of low birthweight and preterm babies would help in the 
understating of prenatal factors influencing the course of pregnancy and, fetal 
growth and development. Such classification tool may serve as an important 
adjunct in epidemiologic research in developing countries. It can also serve as 
a clinical tool for the identification and referral of high risk newborns for 
appropriate care. It may also assist clinicians caring for babies with unknown 
birthweight and gestational age. 

Proposed Course of Project ; Data analysis and manuscript preparation continue 
and are expected to be completed within the next fiscal year. 

Publ ications ; None this year. 



80 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 

NOTICE OF INTTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02518-02 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one tine between ttie borders.) 

Effectiveness of Rapid Plasma Reagin Screening in Gestational Syphilis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: N. Tafari Visiting Scientist EB , DESPR, NICHD 



Other: J.D. Clemens 
M.R. Rao 



Chief 

Visiting Scientist 



EB, DESPR, NICHD 
EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

National Health Research Institute, Addis Ababa, Ethiopia (D. Zewdie) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 
0.60 



PROFESSIONAL: 

0.60 



CHECK APPROPRIATE BOX(ES) 

B (a) Human subjects 
B (a1) Minors 
B (a2) Interview/s 



D (b) Human tissues 



D (c) Neither 



SUMMARY OF V^ORK (Use standard unreduced type. Do not exceed the space provided.) 

Congenital syphilis (CS) remains a major source of perinatal and infant mortality 
in sub-Saharan Africa and in high risk groups in the U.S. A major reason for the 
continued occurrence of CS is the inability of primary health care (PHC) services 
to provide a sustainable screening and treatment program. Dependence on 
centralized system of screening has been identified as the critical obstacle in the 
performance of the screening program. Moreover, a number of reports from the U.S. 
have drawn attention to tho fact that current recommendations of prenatal treatment 
of gestational syphilis (GS) for prevention of CS may be inadequate. 

As part of the national effort to improve the effectiveness of maternal and child 
health programs, the Ministry of Health of Ethiopia introduced a program of rapid 
plasma reagin "teardrop" (RPR-TD) test for screening and treatment of GS in PHC 
units without direct access to a medical laboratory. The RPR-TD was chosen over 
the standard venereal disease (VDRL) test because of several operational advantages 
including antigen stability and, possibility to carry out the test without the use 
of laboratory equipment such as the water bath, microscope or mechanical rotator. 
The test can be done and treatment can be given at initial contact thereby 
increasing patient and health care provider compliance. While a tendency towards 
reduction in fetal and infant mortality attributable to treatment could be 
demonstrated, there remained a large mortality gradient between the exposed and 
unexposed that appeared to be related in part to treatment failure, to diagnostic 
inaccuracy and to the prec-Jiice of HIV co-infection. 



m: 



PHS 6040 (Rev. 5/92) 



o 



ZOl HD 02518-02 EB 

Project Description ; 

Objectives : The aim of the study was to assess operationally the diagnostic 
performance of the rapid plasma reagin "teardrop" test (RPR-TD) in the prevention 
of congenital syphilis. 

Methods Employed ; The study was undertaken to evaluate a recently introduced 
program of the Ministry of Health of Ethiopia for screening and treatment of 
gestational syphilis (GS). The RPR-TD testing and treatment according to WHO 
recommendations of 2.4 million units of benzathine penicillin (BPG) was 
introduced in eight primary health care facilities offering prenatal care. In 
addition to routine clinical data, information on socioeconomic and demographic 
characteristics were obtained on all women undergoing the screening test. We 
assembled a cohort of 580 women who gave positive test on RPR-TD and who received 
BPG and 580 randomly selected women who were RPR-TD negative at the time of 
screening. The primary outcome measure was infant survival up to the age 12 
weeks. We retrospectively evaluated the diagnostic performance of RPR-TD by 
comparing the results with VDRL microf locculation slide test and fluorescent 
treponemal antibody absorption test (FTA-ABS). All sera tested with VDRL and FTA- 
ABS were also screened for HIV with enzyme linked immunosorbent assays (ELISAs) 
and confirmed by western blotting. 

Major Findings ; Of the 3484 women screened with RPR-TD, 580 (17%) were positive. 
Comparison of the diagnostic performance of screening test with VDRL and FTA-ABS 
test results showed a sensitivity of 66% and specificity of 89%. The prevalence 
of GS according to VDRL/FTA-ABS test was 10% (357/3484). We followed 479 (83%) 
of the RPR-TD positive 513 (88%) of the RPR-TD negative pregnancies. Fetal and 
infant mortality was highest in those with GS/HIV co-infection (20/35 or 57%), 
and lowest in the group with no serological evidence for neither infection 
(17/617 or 5%). HIV infection was associated with 26% mortality and syphilis 
infection with 10% mortality. We postulate three hypotheses to account for 
failure to demonstrate significant reduction in fetal and infant mortality; 
failure to detect infection because of low sensitivity of the screening test, 
reduced efficacy of prenatal BPG therapy and, effect modification by HIV-1 co- 
infection. 

Significance to Biomedical Research and the Program of the Institute ; Syphilis 
and HIV are sources of excess perinatal and early infant mortality both in US 
women with high risk behavior and in African women living in rural poverty. The 
study offers a unique opportunity to measure the separate effects of failure of 
screening, treatment failure and, the effect modification of HIV co-infection on 
fetal and early infant mortality. The results of the study can indicate 
directions of future efforts towards improvement of the efficacy and 
effectiveness of screening and treatment of GS for the prevention of CS. 

Proposed Course of Project ; Analysis of the interaction between HIV and syphilis 
upon fetal and infant mortality will be undertaken in the coming year. 

Publications ; A manuscript has been submitted to the American Journal of Public 
Health. 



O A, 



DEPARTMEhfT OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SEFMCE 

NOTICE OF IMTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02519-01 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (SO characters or less. Title must fit on one line between the borders.) 

Determination of Protection Level of Maternal Antibody to Group B Streptococcus 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: F.-y. Lin Research Medical Officer EB, DESPR, NICHD 

Others: J.D. Clemens Branch Chief EB, DESPR, NICHD 

R.A. Brenner Senior Staff Fellow EB, DESPR, NICHD 

Y.R. Johnson IRTA Fellow EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

University of Alabama (B.Gray); Baylor College of Medicine (L.Weisman); Columbia 
University (J.Regan); Univ-ersity of Florida (P.Clark); UMDNJ (G.G .Rhoads ) ; Magee 
Womens Hospital (B . Brozanski ) : Oakland Children's Hospital (P.Azimi^ 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



1.2 



PROFESSIONAL: 



1.2 



CHECK APPROPRIATE BOX(ES) 

H (a) Human subjects 
B (a1) Minors 
B (a2) Interview's 



B (b) Human tissues 



n (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Group B Streptococcus (GET)) is the leading cause of neonatal sepsis and meningitis. 
Neonatal GBS diseases occur in two forms: early-onset and late-onset. Early-onset 
neonatal GBS disease occurs within 7 days of life, and the late-onset disease 
occurs after 7 days of life. Prevention of neonatal GBS through vaccination 
therefore could only be achieved through maternal transfer of antibodies to newborn 
infants. GBS capsular polysaccharide (la, lb, II, III, IV, V) is a virulent factor 
and protective antigen. Vaccine based on capsular polysaccharide and protein 
conjugation is being developed at NICHD as well as other laboratories. 
Determination of a protective threshold would provide an important target for which 
candidate GBS vaccines administered during pregnancy should aim. 

The major objective of the study is to evaluate, for each GBS serotype, the pattern 
of the protective association between naturally acquired, serotype-homologous 
maternal serum IgG antibody titers and the risk of development of early-onset GBS 
disease. 

The surveillance began as of July 1995. The target is to accrue 140 early-onset 
GBS cases and 1120 controls (colonized infants) during the 30 month study. 



PHS 6040 (Rev. 5/92) 



Cj< 



ZOl HD 02519-01 EB 
Pro.ject Description 
Ob.iectives ; 

1) The objective of this study is to evaluate the protective relationship between 
titers of maternal antibodies at delivery and the risk of Early-onset Group B 
Streptococcal (GBS) disease caused by GBS serotypes la, lb, II, III, and V. 

2) A secondary objective is to explore the relationship between GBS antibody 
titers in umbilical cord sera and the risk of early-onset disease, to assess the 
modifying role of placental transfer of antibodies. 

Methods Employed : Two groups of infants will be recruited in this study: infants 
who have developed invasive GBS disease ("cases") and infants who have not 
developed disease despite being surface colonized ("controls"). This study will 
be conducted at 13 hospitals affiliated with 7 U.S. academic institutions. 

Cases will be identified through active surveillance procedures established at 
each of the study sites. Contemporaneous controls will be assembled via surface 
cultures obtained from the throat, anus, umbilicus and ear canal on a systematic 
sample of infants born at these same sites. 

Maternal and cord sera will be collected from these infants and their mothers. 
GBS isolates from case infants and from control mothers will be serotyped. 
Titers of IgG anti-CP antibodies to the infecting GBS serotype will be measured 
in maternal and cord sera, using a standardized assay. To characterize 
potentially confounding variables, patient charts will be reviewed. Cases and 
controls will be contrasted for the presence of antibody at various titers, both 
in simple analyses and in multivariable analyses that control for potentially 
confounding variables. 

Major Findings : None yet. 

Significance to Biomedical Research and the Program of the Institute : 
Establishment of a protective threshold of maternal antibody to serotype-specif ic 
polysaccharide of GBS will provide an important target for which candidate GBS 
vaccines (a vaccine that is being developed in the Institute) administered during 
pregnancy should aim. 

Proposed Course of Project : The surveillance began in July 1995 and will 
continue through January of 1998. 

Publ ications : None yet. 

Contract Report : The study is conducted in collaboration with the University of 
Alabama in Birmingham, Baylor College of Medicine, Columbia University, 
University of Florida, Magee Womens Hospital of University of Pittsburgh, New 
Jersey School of Medicine and Dentistry and Children's Hospital Medical Center 
of Northern California under the following contracts: NOl-HD-4-3214, NOl-HD-4- 
3215, NOl-HD-4-3216, NOl-HD-4-3217, NOl-HD-4-3218, NOl-HD-4-3219, and NOl-HD-4- 
3220. This project is supported by Westat that serves as a data coordinating 



S' 



ZOl HD 02519-01 EB 

center under contract number NOl-HD-5-3233. The 7 clinical centers are 
contracted to enroll GBS cases and colonized control infants. During the current 
fiscal year, a study protocol, a manual of procedure, and data forms were 
developed and the study enrollment began. During FY95 $1,179,603 has been 
allotted to this project. 



61 



kj 



DGPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SEFWICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02520-01 EB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 chamctem or less. Title must fit on one line between the borders.) 

Maternal Caffeine Use in Pregnancy Outcome 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: M.A. Klebanoff Research Medical Officer 

Others: Richard Levine Research Medical Officer 

John Clemens Chief, Epidemiology Branch 

Rebecca DerSimonian Statistician 



EB, DESPR, NICHD 

EB, DESPR, NICHD 

EB, DESPR, NICHD 

BMSB, DESPR, NICHD 



COOPERATING UNITS (if any) 

Center for Human Toxicology, University of Utah (D.Rollins) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.15 



PROFESSIONAL: 



0.15 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



D (b) Human tissues 



B (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The role of maternal caffeine consumption in the pathogenesis of adverse pregnancy 
outcomes in controversial. Several studies have found that women who consume 
caffeine are at increased risk of spontaneous abortion and fetal growth retardation 
compared to non users. However, other, equally well-done studies have found no 
harmful effects of caffeine consumption. In addition, several studies have 
reported that caffeine is harmful only among women who smoke. 

All previous studies of this question have relied on maternally-reported caffeine 
use; no studies have employed a biomarker for caffeine. This project will first 
validate the use of serum caffeine and its metabolites as a marker for caffeine 
intake, and will then study these serum markers as a risk factor for adverse 
pregnancy outcome. 



PHS 6040 (Rev. 5/92) 



iJV 



ZOl HD 02520-01 EB 
Project Description 
Objectives ; 

1. To determine whether serum levels of caffeine and/or its primary metabolites 
are correlated in a useful manner with caffeine intake. 

2. To determine whether maternal caffeine use during pregnancy, as determined by 
serum levels of caffeine and/or its primary metabolites, is associated with 
spontaneous abortion. 

3. To determine whether maternal caffeine use, as determined by serum levels of 
caffeine and/or its primary metabolites, is associated with reduced fetal growth. 

4. To determine if increasing maternal caffeine consumption during pregnancy as 
determined by serum levels of caffeine and/or its primary metabolites, is 
associated with less favorable neuro-development in the offspring. 

5. To determine if caffeine is more deleterious in smokers compared to non- 
smokers. 

Methods Employed ; In the first phase, the utility of serum caffeine and in 
paraxanthine to serve as a biomarker for caffeine intake will be evaluated. 
Women who completed a 24-hour dietary recall and had blood drawn at an 
unspecified time during working hours will be studied. The women were selected 
based upon their reported smoking and caffeine use. Thirty women were chosen at 
random from within each of 8 strata defined by 1) smoker/non-smoker and 2) 
caffeine consumption of <0.8 mg/kg a day; 0.8-1.49 mg/kg a day; 1.50-2.99 mg/kg 
a day; and >3.0 mg/kg a day. 

Should the pilot data indicate that serum markers are useful, the main study will 
be done. In this study the first serum obtained from women experiencing a 
spontaneous abortion will be compared to serum obtained from women experiencing 
a live birth. The controls will be matched to the cases on age, time in 
pregnancy when the serum was drawn, study center, and smoking status. In 
addition, among controls, the first serum drawn after 26 weeks gestation will be 
evaluated to determine the association between caffeine and fetal growth 
retardation. Final models will include interaction times between caffeine and 
smoking. The exact marker for caffeine intake will depend on the pilot study 
results. 

Major Findings ; None yet. 

Significance to Biomedical Research and the Program of the Institute ; Caffeine 
is the most commonly used drug during pregnancy. Between coffee, tea, chocolate, 
and soft drinks, over half of all pregnant women are exposed to caffeine. If 
caffeine truly is harmful, then pregnant women should be encouraged to cut back 
or quit. If it is not harmful, then women may be reassured that they may safely 
continue its use. If caffeine is harmful only among smokers, then those women 
who do not smoke may be reassured, and those who smoke will have another reason 
to quit smoking. 

Proposed Course of Project ; The serum from the pilot study is currently being 
analyzed. The main study will proceed pending these results. 

Publications : None yet 



DEPAHTMENT OF HEALTH AND HUkMN SEFMCES - PUBLIC HEALTH SEFWICE 

NOTICE OF im"RAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02521-01 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Replacement of Pregnancy Losses: Interpregnancy Interval in Adolescence 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Y.R. Johnson IRTA Fellow EB, DESPR, NICHD 

Others: K.F. Yu Statistician BMSB, DESPR, NICHD 

P.M. Moyer Computer Specialist CSB, DESPR, NICHD 



COOPERATING UNITS (if any) 

NIDCD (HJ Hoffman) 



LAB/BRANCH 



Epidemiology Branch 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.5 



PROFESSIONAL: 



0.5 



OTHER: 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



n (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Adolescent age at first pregnancy is one of the single most important risk factors 
for adverse pregnancy outcomes. Adolescent women have higher rates of abortion, 
often this is utilized as a means of contraception. Adolescents also have a higher 
frequency of low birth weight and preterm births, as well as fetal and neonatal 
death(s). The important risk factors which have been shown to be associated with 
poor pregnancy outcomes in adolescents include biological and behavioral factors: 
inadequate prenatal care, maternal smoking, maternal drug and alcohol use, poor 
pregravid nutritional status, acute/chronic medical conditions, inadequate 
education, as well as shortened spacing (<two years) between successive 
pregnancies . 

Shortened interpregnancy intervals increase the risk of adverse pregnancy outcomes. 
The association between birth spacing and pregnancy outcome may be related to 
biological factors such as, decreased uterine tonicity, depleted maternal 
nutritional reserves, and inadequate time to achieve physiological equilibrium 
prior to the next conception. One phenomenon which occurs in older women of 
reproductive age, and is associated with shortened interpregnancy intervals is 
reproductive compensation. This occurs as a result of perinatal loss in the 
previous pregnancy and the desire for women to replace that loss sooner. 

The purpose of this study is to evaluate the relationship between prior perinatal 
loss (abortion, fetal and neonatal death) and the time between successive 
pregnancies in adolescent women. This objective will be achieved using a 
population-based analysis. An adolescent cohort was assembled using the Missouri 
linked data set of live births, late fetal and infant deaths from 1978 through 
1990. 



PHS 6040 (Rev. 5/92) 



6u 



ZOl HD 02521-01 EB 

Pro.ject Description 

Ob.jectives : To evaluate the relationship between prior perinatal loss (abortion, 
fetal and neonatal deaths) and time between successive pregnancies in a cohort 
of adolescent women. 

Methods Employed : An adolescent cohort of women was assembled from the Missouri 
linked data set of live births, late fetal and infant deaths from 1978 through 
1990. Maternal and infant sibships were organized by a computerized linkage 
using maternal name, race, age, and parity. Higher-order births identified were 
linked to the previous birth by year/month of last birth/fetal death. Seven 
adolescent cohorts' ages <13-19 years were organized based on age at first 
pregnancy. Kaplan-Meier survival analysis was used for each age cohort as a 
function of interpregnancy interval. Age cohorts were compared with regard to 
time to subsequent pregnancy for up to three pregnancy events. Statistical 
significance of survival curve comparisons between each age cohort and a 24-29 
year old reference group was based on the log rank chi square test. Cox 
regression analysis was employed to compare the hazards based on the outcome of 
the previous pregnancy controlling for race, maternal education, marital status, 
maternal smoking, pregravid body mass index (BMI), maternal medical risk factors, 
and prenatal care utilization. Interpregnancy interval was defined as the time 
from the previous birth to the next conception (defined by last menstrual 
period). 

Major Findings : Preliminary results show: 

1) The likelihood of a second pregnancy within one year of the first was greater 
for the youngest adolescents (up to 12%) compared to the reference group; 

2) Women who began reproduction as adolescents were more likely to have higher 
parity (40-65% have at least three pregnancies) compared to the reference group; 

3) The rates of abortion were greater for adolescents (30-50%) compared to the 
reference group (9-11%); 

4) The risk of a subsequent pregnancy was 2.5 fold higher in adolescent women 
with a previous pregnancy loss compared to women with a previous term live birth; 

5) Adolescent women tend to have shortened interpregnancy intervals (<1.5 years); 

6) Among adolescent women, a prior pregnancy loss was associated with up to a 
twofold risk of a subsequent pregnancy within 1.5 years compared to women with 
a previous term live birth. 

Significance to Biomedical Research and the Program of the Institute : This study 
will enhance our understanding of the phenomenon of reproductive compensation in 
adolescent women as a factor contributing to shortened spacing between successive 
births. This information will have a significant public health impact with 
regard to counseling adolescent women about proper birth spacing to decrease the 
risk of adverse pregnancy outcomes in this high risk population. 



tuJ 



ZOl HD 02521-01 EB 

Proposed Course of Pro.iect : Completion of the final computer analysis and 
manuscript preparation will require an additional three to four months. 

Publications: None 



ou 



DEPAFITMENT OF HEALTH AND HUMAN SERVJCES - PUBLIC HEALTH SERVICE 

NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02522-01 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Intrapartum Antibiotic Prophylaxis against Early-Onset Grp.B Streptococcal Disease 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Y.R. Johnson IRTA Fellow EB, DESPR, NICHD 

Others: J.D. Clemens Branch Chief EB, DESPR, NICHD 

R.A. Brenner Senior Staff Fellow EB, DESPR, NICHD 

F.-Y. Lin Research Medical Officer EB, DESPR, NICHD 



COOPERATING UNITS (if any) 

University of Alabama (BM Gray, JB Philips); Baylor College of Medicine (LE 
Weisman) ; Columbia Univ. ( JA Regan); Univ. of Florida (P Clark); Univ. of Pittsburgh 
(BS Brozanski. RL Sweet); Univ. Med .Dent .NJ (GG Rhoads ) : Med. Ctr .No .Calif . (PH Azimi) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.4 



PROFESSIONAL: 



0.4 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (al) Minors 
n (a2) Interviews 



D (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Neonatal group B streptococcal (GBS) disease is the most common disease affecting 
newborn infants with an estimated incidence of 1.8 per 1000 live births. Twenty- 
five percent of neonatal GBS disease occurs in premature infants. GBS disease 
results in significant mortality and morbidity in newborns. Colonization of the 
infant occurs via vertical transmission from a colonized mother. Among pregnant 
women, the rate of GBS colonization is approximately 20-30Z. Approximately 42-722 
of infants born to colonized mothers will become colonized, and 1-2Z of these 
infants will develop invasive GBS disease. A major factor which may modify the 
rate of colonization, and possibly the risk of invasive disease is intrapartum 
antibiotic prophylaxis given to women who are at definably high risk for early- 
onset GBS disease. This intervention strategy has been shown to be near absolute 
in efficacy trials in conferring protection; it seems probable that substantially 
lower levels of protection occur when recommended regimens are attempted in routine 
clinical practice. Supporting this notion is data indicating that intrapartum 
antibiotic prophylaxis fails to prevent up to 25-30Z of cases of early-onset 
neonatal GBS disease. The purpose of this retrospective case-control study is to 
evaluate the effectiveness of intrapartum antibiotic prophylaxis given to high risk 
women in routine clinical obstetric practice with the intent to prevent early-onset 
neonatal GBS disease. This objective will be accomplished by the following: 1) 
assembled case and control infants will comprise all live births born to high risk 
women at the participating hospitals over three years from January 1, 1992 through 
December 31, 1994; 2) cases and controls will be assembled from a retrospective 
review of hospital laboratory logs; and 3) perform detailed chart abstraction of 
clinical and demographic data from the medical records of all eligible case-control 
maternal and infant pairs. 



PHS 6040 (Rev. 5/92) 



y.. 



ZOl HD 02522-01 EB 

Project Description 

Objectives : To assess the protection associated with the administration of 
antibiotics given during labor to high risk pregnant women as prophylaxis to 
prevent early-onset neonatal group B streptococcal disease (GBS), 

Methods Employed ; This is a retrospective case-control study designed to 
evaluate the effectiveness of intraprtum antibiotic prophylaxis given to high 
risk women in routine clinical obstetric practice with the intent to prevent 
early-onset neonatal GBS disease. Cases and controls will be assembled via 
retrospective review of hospital laboratory logs, hospital discharge diagnoses, 
and obstetric delivery logs. The sampling frame will comprise all live births 
occurring at the participating study sites between January 1, 1992 through 
December 31, 1994; infants born to high risk mothers will be potentially eligible 
for enrollment in the study. Case infants will be defined as those with 
documented early-onset GBS disease, and control infants will be defined as those 
with no documented early-onset GBS disease. Two Controls will be matched to each 
case based on the hospital of birth, date of birth within three months, and 
gestational age within two weeks. After assessment of eligibility for enrollment 
in the study, detailed chart abstraction of clinical and demographic data from 
the medical records of case and control maternal-infant pairs will take place. 
Chart abstraction forms designed for this study will be completed on all eligible 
case and control infants at each study site. These data will be sent to NICHD 
for data entry, data editing, and analysis. 

Major Findings ; None to date (project is under development). 

Significance to Biomedical Research and the Program of the Institute ; This study 
will greatly enhance our understanding of the effectiveness of intrapartum 
antibiotic prophylaxis intended to prevent early-onset GBS disease in newborn 
infants. Results from this study will allow better recommendations to be made 
regarding treatment of mothers and their newborns. In addition, this study of 
the effectiveness of intrapartum antibiotic prophylaxis will greatly assist in 
the proper interpretation of the protection conferred by maternal serum 
antibodies against GBS disease, an NIH sponsored study currently taking place at 
seven participating U.S. academic centers. 

Proposed Course of Project : Three to four months will be required for data 
collection, and an additional two months for data entry, editing, and analysis. 

Publications: None 



9.: 



DEPAFJTMEhn" OF HEALTH AND HUMAN SEFMCES - PUBLIC HEALTH SEFMCE 

NOTICE OF INTTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02523-01 EB 



PERIOD COVERED 

October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Studies Based on Maternally Linked Birth Registries of Missouri and Utah 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: A. A. Herman Visiting Scientist EB, DESPR, NICHD 



Others ; 



K.F. Yu 
Y.R. Johnson 



Mathematical Statistician 
IRTA Fellow 



BMSB, DESPR, NICHD 
EB, DESPR, NICHD 



COOPEFIATING UNITS (if any) 

NIDCD (H.Hoffman); Missouri Department of Health (G.Land, J . Stockbauer , W. 
Schramm); University of Utah (R.Ward, A. Eraser) 



LABmRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 

0.2 



PROFESSIONAL 

0.2 



CHECK APPROPRIATE BOX(ES) 

H (a) Human subjects 
H (a1) Minors 
a (a2) Interviews 



B (b) Human tissues 



G (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. 



T exceed the space provided.) 



These studies focus on: 1) the impact of adolescent age at first pregnancy on later 
pregnancy outcome; 2) the relation between interpregnancy interval and later 
pregnancy outcomes; 3) the risk of repeating poor pregnancy outcomes. The data 
sets are unique in that all pregnancies of women from Missouri and Utah are linked 
into sibships. These data allow the analyst to examine longitudinal pregnancy 
data. 



PHS 6040 (Rev. 5/92) 



^3 



o 



ZOIHD 02523-01 EB 

Pro.iect Description : 

Objectives : 1) To examine the impact of adolescent age at first pregnancy and 
age greater that 35 years at first pregnancy on later pregnancy outcome; 2) the 
relation between interpregnancy interval and later pregnancy outcomes; 3) the 
risk of repeating poor pregnancy outcomes. The data sets are unique in that all 
pregnancies of women from Missouri and Utah are linked into sibships. 

Methods Employed : Analytic methods include risk-based analysis of recurrent 
mortality and morbidity, life-table analyses and cox regression analysis, 
indirect standardization of mortality rates by birth weight and gestational age, 
examination of bivariate distributions using contour diagrams, fuzzy clustering 
of growth retarded and non growth retarded infants, non-parametric logistic 
regression, and other procedures applicable to population-based data. The 
epidemiologic approach is that of a "prospective" cohort design, which permits 
analyses by case-control, cross-sectional or longitudinal means. 

Ma.ior Findings ; Abstracts dealing with interpregnancy interval among adolescents 
(Society for Pediatric Research and Society for Pediatric Epidemiologic 
Research), increasing risk for perinatal mortality, preterm birth and 
intrauterine growth retardation among adolescents (Society for Pediatric 
Epidemiologic Research), sociodemographic profiles and adverse reproductive 
outcome (American Public Health Association), the recurrence of birth defects 
(American Society of Human Genetics), and growth retardation and SIDS have been 
presented in 1995. These results showed that: 1) adolescent women tend to have 
shorter interpregnancy intervals, 2) the shorter intervals have an adverse effect 
on pregnancy outcomes, 3) adolescents become pregnant more frequently than older 
women, 4) risks for perinatal mortality, preterm birth and growth retardation 
increases with parity among teenagers, 5) adolescents at increased risk for these 
outcomes have higher rates of poor pregnancy outcome than older women with 
comparable risk factors, 6) siblings of SIDS infants were not at increased risk 
for lUGR when maternal smoking was accounted for, 7) both similar and dissimilar 
birth defects recur with a statistically significant increased frequency, but 
context and paternity do not further increase the risk, and 8) paternal low SES 
had a greater influence on low birth weight than maternal low SES. The impact of 
low SES could not be explained solely by the frequency of high risk behavior 
among low income families. 

Significance to Biomedical Research and the Program of the Institute : These 
studies will allow researchers to more fully explore the longitudinal impact of 
risk factors on pregnancy outcome. The repeater studies are among the first using 
United States data. Early interventions among high risk women may eventually lead 
to a reduction of preterm birth and lUGR. 

Proposed Course of Pro.iect : The initial year of data analysis has been fruitful. 
Dr. Herman will continue to examine the longitudinal impact of risk factors, 
recurrence risk, the impact of low SES on pregnancy and the impact of early - or 
late first pregnancy on SES. 

Publications : None yet. 






DEPAffTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICe 

NOTICE OF im"RAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02524-01 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Occupational Nonfatal Injuries in U.S. Children 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 



Other: K.A. Dunn 



IRTA Fellow 



EB, DESPR, NICHD 



COOPERATINQ UNITS (if any) 

U.S. Bureau of the Census (E. Lamas) 



LAB/BRANCH 



Epidemiology Branch 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda, Maryland 20892 



TOTAL STAFF YEARS: 



0.3 



PROFESSIONAL: 



0.3 



OTHER: 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
n (a2) Interviews 



D (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

As many as 300 children a year may be killed while working in the U.S.; an unknown 
number suffer nonfatal injuries on the job. Current information on occupational 
injuries in children is based entirely on death certificates or workmen's 
compensation reporting. Both sources are known to contain extensive and biased 
underreporting for children. Many children are working illegally, "of f-the-books" , 
or in hazardous conditions including, but not limited to, those inappropriate for 
the child's health and developmental status. Determination of high risk 
occupations and incidence and severity of non-fatal injuries has been made a 
priority in the injury control and pediatric emergency treatment communities. 
Current policy questions on effects of 'school-to-work' programs require supporting 
baseline data on current work practices and injuries. NICHD staff are working 
collaboratively across Federal Agencies to determine the incidence of non-fatal 
injuries occurring to children in the workplace by occupational category exposure 
time. Dr. Dunn is completing a study of occupational exposure time for adolescents 
ages 15-18 from the Census Bureau Survey of Income and Program Productivity (SIPP). 
Her work will be used to provide unique and relatively unbiased exposure data for 
future epidemiological studies. Dr. Overpeck has prepared injury questions to be 
fielded in the SIPP in 1995 and later years. This will enable researchers to 
target interventions effectively by identifying both high risk children and high 
risk occupational categories. 



PHS 6040 (Rev. 5/92) 



C?Q 



ZOl HD 02524-01 EB 

Project Description : Research on adolescent occupational injury in the U.S. has 
been planned in three phases using the Survey of Income and Program Participation 
(SIPP). Exposure data for working adolescents is under development now by Dr. 
Kathleen Dunn. Questions on injuries to adolescents will be added to the SIPP 
for the current survey in the field and a new survey under development. The goal 
is to determine injury incidence and enable researchers to target interventions 
effectively by identifying both high risk children and high risk occupational 
categories. 

Methods Employed ; The (SIPP) is a continuous household survey fielded by the 
U.S. Census Bureau. A household is followed for 2 ^ years. Core questions and 
special topical supplements are administered at four month intervals. SIPP 
currently determines occupational status, category and time for all adults >15 
years and tracks those who move. Limited injury questions have been included in 
the past. Dr. Overpeck has developed questions on injuries at ages 12-17 that 
will be included for a four-month period of 1995 with a Child Well-being Module 
sponsored by NICHD' s Demographic and Behavioral Science Branch. She will also 
have occupational injury questions included for 50,000 households in the survey 
to begin in 1996. Injuries will be assessed for medical treatment and severity. 
Analysis through multivariate regression techniques appropriate for longitudinal 
complex multistage surveys will be performed. 

Major Findinqs : None yet, 

Siqnificance to Biomedical Research and the Proqram of the Institute ; As many 
as 300 children a year may be killed while working; an unknown number suffer 
nonfatal injuries on the job. Current information on occupational injuries in 
children is based entirely on death certificates or workmen's compensation 
reporting. Both sources are known to contain extensive and biased underreporting 
for children. Many children are working illegally, "off-the-books" , or in 
hazardous conditions including, but not limited to, those inappropriate for the 
child's health and developmental status. Determination of high risk occupations 
and incidence and severity of non-fatal injuries has been made a priority in the 
injury control and pediatric emergency treatment communities. NICHD staff are 
working collaboratively across Federal Agencies to determine the incidence and 
severity of non-fatal injuries occurring to children in the workplace by 
occupational category exposure time. The ultimate goal is to enable researchers 
to target interventions effectively by identifying both high risk children and 
high risk occupational categories. 

Proposed Course ; Dr. Dunn's work will be used as part of her dissertation for 
a degree in Epidemiology at the University of North Carolina and will be 
submitted for publication. Dr. Overpeck will use the preliminary work on 
exposures prepared by Dr. Dunn to evaluate the injury data that will be available 
for the first time in late 1996. 

Publications; None to date 



BG 



DEPAF^rME^^■ of health and human sswices - public health sefmce 
NOTICE OF l^^"RAMUFW. RESEARCH PROJECT 



PROJECT NUMBER 



ZOl HD 02525-01 EB 



PERIOD COVERED 



October 1, 1994 through September 30, 1995 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

World Health Organization Study of Health Behavior in School Children (WHO-HBSC) 



PRINCIPAL INVESTIGATOR {List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: M.D. Overpeck Epidemiologist EB, DESPR, NICHD 

Others: N.Krasnegor Chief, HLBB HLBB, CRMC, NICHD 

P.C. Scheldt IPA HLBB, CRMC, NICHD 



COOPERATING UNITS (if any) 

Substance Abuse and Mental Health Services Admin. (B. Rouse); Bar Ilan University, 
Israel (Y.Harel); WHO-HBSC Study Group (27 countries) 



lab/branch 



Epidemiology Branch 



SECTION 



INSTITUTE AND LOCATION 

NICHD, NIH, Bethesda. Maryland 20892 



TOTAL STAFF YEARS; 



0.1 



PROFESSIONAL: 



0.1 



OTHER: 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
n (a2) Interviews 



G (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The WHO coordinates a quadrennial survey that compares health behavior in 
adolescents in 27 countries. The survey is used to monitor and investigate 
patterns, determinants and effects of health related behaviors including smoking, 
substance use, and injury behavior. The U.S. has not participated yet. NICHD 
staff attended meetings with the international working group to learn about the 
process of comparing the data, results of the most recent international studies, 
and to apply to become a collaborating nation. The Substance Abuse and Mental 
Health Services Administration (SAMHSA) has already developed and funded a U.S. 
school-based survey to be fielded in Fall, 1995, that will meet the preliminary 
criteria for U.S. participation in the WHO-HBSC project. The successful completion 
and analysis of the U.S. survey and linkage to the existing data of the ongoing WHO 
surveys will allow the U.S. to compare behavior and related injury factors to data 
from 27 countries of Europe, Canada, and elsewhere. The Epidemiology and Human 
Learning and Behavior Branches of NICHD are collaborating with SAMHSA to conduct 
the data analysis for both the U.S. and international data sets. NICHD will focus 
particularly on injury comparisons. The data will prove invaluable for both 
international and domestic comparisons of injury and related behaviors in 
adolescents to target interventions since injury is the leading cause of death for 
children in the U.S. 



PHS 6040 (Rev. 5/92) 



y? 



ZOl HD 02525-01 EB 

Project Description ; 

Objectives : The World Health Organization Study of Health Behavior in School 
Children (WHO-HBSC)is used to monitor and investigate patterns, determinants and 
effects of health related behaviors including smoking, substance use, and injury 
behavior. The U.S. has not been a participant. Collaboration with the Substance 
Abuse and Mental Health Services Administration (SAMHSA) will permit comparative 
analysis of both the U.S. and international data. NICHD will focus particularly 
on injury and related behaviors. 

Methods Employed : The WHO coordinates a quadrennial survey that compares health 
behavior in adolescents in 27 countries. SAMHSA is collecting comparable data 
in a school-based study of U.S. adolescents. NICHD collaborators in the 
intramural and extramural programs will use descriptive and multivariate 
epidemiologic techniques appropriate for complex multistage complex surveys to 
prepare analytic papers. 

Major Findings : None available yet. 

Significance to Biomedical Research and the Program of the Institute : Injury is 
the leading cause of death for children in the U.S. NICHD has been active in 
promoting injury research as an emerging new endeavor through behavioral and 
epidemiological analyses and will emphasize this area in the collaborative 
analyses. The U.S. has learned much about risk factors and health outcomes 
through such international comparisons on infant mortality, aging, heart disease 
and health behaviors, for example. These data will prove invaluable for both 
international and domestic comparisons of injury and related behaviors in 
adolescents to target interventions appropriate to the U.S. 

Proposed Course : NICHD has proposed joint funding by the intramural and 
extramural programs to support analysis of the SAMHSA survey data. The 
collaborative research team will complete the work through an interagency 
agreement with SAMHSA which has an on-going contract to perform the survey and 
complete the analysis. 

Publications: None to date 



•98 



c- 
















f \_ ^^ ^ 






■^ 













"^^ 
%, 



*>. ,^-<^ *!^X X<^ ■*>, 



/ 



./ 




■^ rf^ \ »»^ ^^ 

*. # .^ \ y* 



<^«> 



=*>, 



^Oto' 



-3^, 



./ 






Research. 
Aiiia2ing Help. 



http://nihlibrary.nih.gov 












^o. 



2 



V 






1 Center Drive 

Bethesda, MD 20892-1 150 

301-496-1080 









YH^ 



# 



# 



^<S'/ 



\ 



/I 



■^ 

y ^ 



HK 






^*/ \^/ \#/ \^/ ve% 



V 



.v*' 



^/-' 11/a 

II T 



^" o^^>. ^S 









# 










4^